Due to its specific characteristics, such as ma-ternal inheritance and absence of recombination, each mtDNA belongs to certain monophyletic clade in the rooted mtDNA tree (haplogroup) according to the mutations it har...Due to its specific characteristics, such as ma-ternal inheritance and absence of recombination, each mtDNA belongs to certain monophyletic clade in the rooted mtDNA tree (haplogroup) according to the mutations it harbors. Rare mutation (excluding parallel mutation) occur-ring at multiple times in different haplogroups could thus be a potential reading error according to the mtDNA phylogeny. This experience has been widely used in double-checking the credibility of the rare mutations in human mtDNA sequences. However, no test has been performed so far for the feasibility of applying this strategy to the rare insertion/deletion (indel) events in mtDNA sequences. In this study, we attempted to relate the rare indels in mtDNAs to their haplogroup status in a total of 2352 individuals from 50 populations in China. Our results show that the insertion of A at position 16259 is restricted to a subclade of haplogroup C and can be verified. The other indel polymorphisms, which occur in the repeat of the deleted or inserted nucleotide(s), may not be distin-guished from phantom mutations from a phylogenetic point of view. Independently and multiply sequencing the frag-ment with the indel is the best and the most reliable way for confirmation.展开更多
Functional deficiency of mismatch repair(MMR) system is one of the mechanisms of tumorigenesis.With the development of the investigation and the requirement from the clinical diagnosis and treatment it is necessary to...Functional deficiency of mismatch repair(MMR) system is one of the mechanisms of tumorigenesis.With the development of the investigation and the requirement from the clinical diagnosis and treatment it is necessary to build up a method to evaluate the functional status of the whole MMR system in the concerned tumors. The original ssDNA and dsDNA from wild type (wt) bacteriophage M13mp2 and its three derivates with mutation points in the lacZa gene have been used to construct two kinds of heteroduplex DNA molecules. One named del(2) has two bases deleted in the negative strand, the other has a G-G mismatch base pair in the negative strand too. Introducing this heteroduplex DNA into E. coli NR9162 (routS^-) without the MMR ability on the indicator plate with x-gal and IPTG,there are three kinds of plaques, mixture plaque as the charaeteristie phenotype of heteroduplex DNA, blue and clearplaques. If the cell extract is mismatch repair competent the percentage of the mixture plaque will decrease after incubation with these heteroduplex DNA, the repair efficiency is expressed in percentage as 100x (1 minus the ratio of percentages of mixture plaque obtained from the extract-treated sample and untreated samples), which can imply the functionai status of MMR system of certain samples. After large T-antigen-dependent SV-40 DNA replication assay cell extract from TK6, a human lymphoblastoid B-cell lymphoma cell line with MMR ability, and Lovo, a human colonic carcinoma cell line with MMR deficiency have incubated with these heteroduplex DNA. The repair efficiency of TK6 to del(2) is more than 60%, to G-G is more than 50%. The Lovo efficiency to del(2) is less than 10%, to G-G is less than 20%.Therefore, in this in vitro model used for functional analysis of mismatch repair of heteroduplex DNA as the repair target,TK6 can serve as the control for MMR proficiency and Lovo as the control for MMR deficiency. Using this model the tumor tissue from a case of hereditary nonpolyposis colorectal cancer (microsatellite instability high, MSI-H) was measured and lack of MMR ability was shown. And a case of sporadic rectal cancer (SRC) (mierosatellite stability, MSS) maintains MMR proficiency. The results indicate that the model is sensitive and dependable. It could be used to measure the funetion status of MMR system in tumor cell and/or tissues. This is a reliable method to investigate the mechanic of tumorigenesis. It is meaningful in the observation of the role of MMR in the initiation and progression of concerned tumors.展开更多
文摘Due to its specific characteristics, such as ma-ternal inheritance and absence of recombination, each mtDNA belongs to certain monophyletic clade in the rooted mtDNA tree (haplogroup) according to the mutations it harbors. Rare mutation (excluding parallel mutation) occur-ring at multiple times in different haplogroups could thus be a potential reading error according to the mtDNA phylogeny. This experience has been widely used in double-checking the credibility of the rare mutations in human mtDNA sequences. However, no test has been performed so far for the feasibility of applying this strategy to the rare insertion/deletion (indel) events in mtDNA sequences. In this study, we attempted to relate the rare indels in mtDNAs to their haplogroup status in a total of 2352 individuals from 50 populations in China. Our results show that the insertion of A at position 16259 is restricted to a subclade of haplogroup C and can be verified. The other indel polymorphisms, which occur in the repeat of the deleted or inserted nucleotide(s), may not be distin-guished from phantom mutations from a phylogenetic point of view. Independently and multiply sequencing the frag-ment with the indel is the best and the most reliable way for confirmation.
文摘Functional deficiency of mismatch repair(MMR) system is one of the mechanisms of tumorigenesis.With the development of the investigation and the requirement from the clinical diagnosis and treatment it is necessary to build up a method to evaluate the functional status of the whole MMR system in the concerned tumors. The original ssDNA and dsDNA from wild type (wt) bacteriophage M13mp2 and its three derivates with mutation points in the lacZa gene have been used to construct two kinds of heteroduplex DNA molecules. One named del(2) has two bases deleted in the negative strand, the other has a G-G mismatch base pair in the negative strand too. Introducing this heteroduplex DNA into E. coli NR9162 (routS^-) without the MMR ability on the indicator plate with x-gal and IPTG,there are three kinds of plaques, mixture plaque as the charaeteristie phenotype of heteroduplex DNA, blue and clearplaques. If the cell extract is mismatch repair competent the percentage of the mixture plaque will decrease after incubation with these heteroduplex DNA, the repair efficiency is expressed in percentage as 100x (1 minus the ratio of percentages of mixture plaque obtained from the extract-treated sample and untreated samples), which can imply the functionai status of MMR system of certain samples. After large T-antigen-dependent SV-40 DNA replication assay cell extract from TK6, a human lymphoblastoid B-cell lymphoma cell line with MMR ability, and Lovo, a human colonic carcinoma cell line with MMR deficiency have incubated with these heteroduplex DNA. The repair efficiency of TK6 to del(2) is more than 60%, to G-G is more than 50%. The Lovo efficiency to del(2) is less than 10%, to G-G is less than 20%.Therefore, in this in vitro model used for functional analysis of mismatch repair of heteroduplex DNA as the repair target,TK6 can serve as the control for MMR proficiency and Lovo as the control for MMR deficiency. Using this model the tumor tissue from a case of hereditary nonpolyposis colorectal cancer (microsatellite instability high, MSI-H) was measured and lack of MMR ability was shown. And a case of sporadic rectal cancer (SRC) (mierosatellite stability, MSS) maintains MMR proficiency. The results indicate that the model is sensitive and dependable. It could be used to measure the funetion status of MMR system in tumor cell and/or tissues. This is a reliable method to investigate the mechanic of tumorigenesis. It is meaningful in the observation of the role of MMR in the initiation and progression of concerned tumors.
