Objective: To investigate the relationship betwhen production of carcinoembryonic antigen (CEA) in thehuman cells of different tissues and scquence variation of t CEA promoter in the cellular genomes. and evaluate po-...Objective: To investigate the relationship betwhen production of carcinoembryonic antigen (CEA) in thehuman cells of different tissues and scquence variation of t CEA promoter in the cellular genomes. and evaluate po-tential application of the ets active scqucnce of CEA gene in colorectal carcinoma tissie-specific gene therapy.Methods: Nested-polymerase chain reaction (PCR) was employed to amplify the CEA promoter sequences fromthe genome of human colorectal carcinoma. normal adjacent colonic mucosa tissues. normal blood cells. placentatissues and some established cell lines of human origin. PCR-Southern bloting assay was utilized to define the reliability of the amplified sequences. The scquence variations were evaluated by means of PCR-single stranded conformation polymorphism (PCR-SSCP) and DNA sequencing. Binding effect of the amplified fragment was examinedby Band-Shift assay. Results: No scqucnce variation was found between-135bp and+69bp from the transcription-al initiation site. which was considered to be a core region of CEA promoter. There was no correlativity betweenproduction levels of CEA and sequence variations of CEA gene promoter core region. The fragment amplified either from normal tissues or from colorectal carcinoma tissues could equally bind with the nuclear extract from Lo-Vo cells. Conclusion: Differential level of CEA gene transcription in the colorectal carcinoma cells. as comparedwith normal tissues, was proved not to be related to the sequence variation of CEA promoter core region. CEApromoters, either from normal cellular genome or neoplasm cellular genome. were found suitable for colorectalcarcinoma tissue-specific gene therapy.展开更多
Abstract Leaves of melon were collected and extracted by the CTAB method for total DNA which was used for PCR amplification, obtaining the gene sequence of cucumisin promoter. The sequence results were processed and a...Abstract Leaves of melon were collected and extracted by the CTAB method for total DNA which was used for PCR amplification, obtaining the gene sequence of cucumisin promoter. The sequence results were processed and analyzed with DNAman, DNAstar and other softwares, and bioinformatic element analysis was performed with PlantCARE and PLACE. The analysis results showed that the cucumisin promoter shared 100%, 99% and 99% homology with AY055805, LN713264 and LN681897, respectively. The promoter sequence contains a variety of c/s-acting elements common in fruit promoters of higher plants such as TATA-Box and CAAT-Box, and light-responsive elements, some of which involved in ABA and VP1 responsiveness and salicylic acid responsiveness. This study provides a scien- tific basis for further research on genetic engineering of fruits.展开更多
O6-methylguanine DNA methyltransferase(MGMT), a DNA repair enzyme, has been reported in some congenital malformations, but it is less frequently reported in neural tube defects. This study investigated MGMT mRNA expre...O6-methylguanine DNA methyltransferase(MGMT), a DNA repair enzyme, has been reported in some congenital malformations, but it is less frequently reported in neural tube defects. This study investigated MGMT mRNA expression and methylation levels in the early embryo and in different embryonic stages, as well as the relationship between MGMT and neural tube defects. Spina bifida aperta was induced in rats by a single intragastric administration of all-trans retinoic acid on embryonic day(E) 10, whereas normal control rats received the same amount of olive oil on the same embryonic day. DNA damage was assessed by detecting γ-H2 A.X in spina bifida aperta rats. Real time-polymerase chain reaction was used to examine mRNA expression of MGMT in normal control and spina bifida aperta rats. In normal controls, the MGMT mRNA expression decreased with increasing embryonic days, and was remarkably reduced from E11 to E14, reaching a minimum at E18. In the spina bifida aperta model, γ-H2 A.X protein expression was increased, and mRNA expression of MGMT was markedly decreased on E14, E16, and E18. Bisulfite sequencing polymerase chain reaction for MGMT promoter methylation demonstrated that almost all CpG sites in the MGMT promoter remained unmethylated in both spina bifida aperta rats and normal controls, and there was no significant difference in methylation level between the two groups on either E14 or E18. Our results show that DNA damage occurs in spina bifida aperta rats. The mRNA expression of MGMT is downregulated, and this downregulation is independent of promoter DNA methylation.展开更多
AIM: The GFAP was traditionally considered to be a biomarker for neural gila (mainly astrocytes and nonmyelinating Schwann cells). Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astr...AIM: The GFAP was traditionally considered to be a biomarker for neural gila (mainly astrocytes and nonmyelinating Schwann cells). Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astrocytes in vitro and in vivo. More recently, GFAP was also established as one of the several makers for identifying hepatic stellate cells (HSC). In this project, possible application of the same 2.2-kb human GFAP promoter for targeting HSC was investigated. METHODS: The GFAP-lacZ transgene was transfected into various cell lines (HSC, hepatocyte, and other nonHSC cell types). The transgene expression specificity was determined by X-gal staining of the β-galactosidase activity. And the responsiveness of the transgene was tested with a typical pro-fibrotic cytokine TGF-β1. The expression of endogenous GFAP gene was assessed by real-time RT-PCR, providing a reference for the transgene expression. RESULTS: The results demonstrated for the first time that the 2.2 kb hGFAP promoter was not only capable of directing HSC-specific expression, but also responding to a known pro-fibrogenic cytokine TGF-β1 by upregulation in a doseand time-dependent manner, similar to the endogenous GFAP. CONCLUSION: In conclusion, these findings suggested novel utilities for using the GFAP promoter to specifically manipulate HSC for therapeutic purpose.展开更多
The 3.1 kb BamHI fragment containing the chloroplast rbcL gene from grape (Vitis vinifera L) hasbeen cloned and its restriction map and nucleotide sequence determined. The complete nucleotidesequence is 20O4 bp long w...The 3.1 kb BamHI fragment containing the chloroplast rbcL gene from grape (Vitis vinifera L) hasbeen cloned and its restriction map and nucleotide sequence determined. The complete nucleotidesequence is 20O4 bp long with a coding sequence of 1428 bp, which cncode a polypeptide of 475amino acids with a predicted molecular weight of 53 kd. The 5' upstream sequence including theputative promoter is 358 bp, with a -10 sequence (TAAAAT), a -35 sequence (TTGCGC) and theSD sequence (GGAGG). The 218 bp long 3' downstream sequence contains three transcription stem-loop tendnation structures. The homologies of this gene with those of tobacco, petunia, spinach,alfalfa, rice and maise are 91.5%, 91.4% 90.2%, 89.8%, 86.3% and 84.5% respectively while thehomologies between their putative polypeptide sequences are 92.2% 91.6%, 92.2%, 93.7%, 93.5%and 90.1% respectively.展开更多
A 3.2 kb EcoRI DNA fragment containing the complete chloroplast rbcL gene from Acanthopanax brachypus has been cloned. In addition to the 1428 bp coding region encoding 475 amino acids of the gene, 278 bp upstream and...A 3.2 kb EcoRI DNA fragment containing the complete chloroplast rbcL gene from Acanthopanax brachypus has been cloned. In addition to the 1428 bp coding region encoding 475 amino acids of the gene, 278 bp upstream and 218 bp downstream were also sequenced. Possible ctpl and ctp2, equivalent to prokaryotic-35 and -10 elements, were found as TTGCGC and TACAAT respectively. The 5' untranslated leader region is 194 bp and the putative ribosome binding site in this region is GGAGG,located immediately upstream of the start codon. The 3' unrtanslated region contains two inverted repeat sequences, which in the mRNA might form stem-loop structures. The homology of rbcL amino acid sequence between A. brachypus and tobacco, spinach, pea, alfalfa, maize, rice, pine,Marchantia, Chlamydomonas and Anacystis are, respectively 93.05%,94.11%, 94.53%,89.68%, 92.21%, 2.21%, 92.63%, 87.58% and 80.84%.The promoter regions and part of the 5', 3' non-coding regions of rbcL from various plants were compared.展开更多
Background: Antibiotic growth promoters(AGPs) have been used as growth promoters to maintain animal intestinal health and improve feed efficiency in broilers by inhibiting pathogen proliferation. In view of the growin...Background: Antibiotic growth promoters(AGPs) have been used as growth promoters to maintain animal intestinal health and improve feed efficiency in broilers by inhibiting pathogen proliferation. In view of the growing emergence of antibiotic-resistant pathogen strains and drug residue issues, novel treatments are increasingly required. This study aimed to compare two antimicrobial approaches for managing pathogen infection and maintaining animal intestinal health in broilers by supplying Apidaecin Api-PR19 and AGPs over 42 d of a feeding trial.Results: Compared with the broilers that were only fed a corn-soybean basal diet(CON group), supplementation with Api-PR19 and AGP(respectively named the ABP and AGP groups) both increased the feed conversion efficiency. When compared with the AGP group, Api-PR19 supplementation could significantly increase the organ index of the bursa of fabricius and subtype H9 antibody level in broiler chickens. Moreover, when compared with the CON group, the intestinal villus height, intestinal nutrient transport, and intestinal s Ig A content were all increased in the Api-PR19 group, while AGP supplementation was harmful to the intestinal villus height and intestinal nutrient transport. By assessing the antibacterial effect of Api-PR19 and antibiotics in vitro and in vivo, we found that Api-PR19 and antibiotics both inhibited the growth of pathogens, including Escherichia coli and Campylobacter jejuni. Furthermore, by using 16 S r RNA gene sequencing, the beneficial bacteria and microbiota in broilers were not disturbed but improved by apidaecin Api-PR19, including the genera of Eubacterium and Christensenella and the species of uncultured_Eubacterium_sp, Clostridium_asparagiforme, and uncultured_Christensenella_sp, which were positively related to improved intestinal development, absorption, and immune function.Conclusion: Apidaecin Api-PR19 treatment could combat pathogen infection and had little negative impact on beneficial bacteria in the gut compared to antibiotic treatment, subsequently improving intestinal development,absorption, and immune function.展开更多
The biosafety of genetically engineered plants has been of concernment in society and science in recent years. The issue of 35S promoter of CaMV has been contentious because of its wide use in plant genetic engineerin...The biosafety of genetically engineered plants has been of concernment in society and science in recent years. The issue of 35S promoter of CaMV has been contentious because of its wide use in plant genetic engineering. The debate on the safety and potential risks of the 35S promoter will be discussed here. Some of concerns are expressed about the dissemination of antibiotic_resistance genes and vector backbone sequences. Various methods and strategies are currently being developed for the marker gene excision and elimination of vector backbone sequences from transgenic plants. In this review, the CRE/ lox system which could get rid of the marker geens and vector backbone sequences will be discussed in detail. Advances in the research of the safety assessment of genetically modified plants using the CRE/ lox system will also be described.展开更多
文摘Objective: To investigate the relationship betwhen production of carcinoembryonic antigen (CEA) in thehuman cells of different tissues and scquence variation of t CEA promoter in the cellular genomes. and evaluate po-tential application of the ets active scqucnce of CEA gene in colorectal carcinoma tissie-specific gene therapy.Methods: Nested-polymerase chain reaction (PCR) was employed to amplify the CEA promoter sequences fromthe genome of human colorectal carcinoma. normal adjacent colonic mucosa tissues. normal blood cells. placentatissues and some established cell lines of human origin. PCR-Southern bloting assay was utilized to define the reliability of the amplified sequences. The scquence variations were evaluated by means of PCR-single stranded conformation polymorphism (PCR-SSCP) and DNA sequencing. Binding effect of the amplified fragment was examinedby Band-Shift assay. Results: No scqucnce variation was found between-135bp and+69bp from the transcription-al initiation site. which was considered to be a core region of CEA promoter. There was no correlativity betweenproduction levels of CEA and sequence variations of CEA gene promoter core region. The fragment amplified either from normal tissues or from colorectal carcinoma tissues could equally bind with the nuclear extract from Lo-Vo cells. Conclusion: Differential level of CEA gene transcription in the colorectal carcinoma cells. as comparedwith normal tissues, was proved not to be related to the sequence variation of CEA promoter core region. CEApromoters, either from normal cellular genome or neoplasm cellular genome. were found suitable for colorectalcarcinoma tissue-specific gene therapy.
基金Supported by Fund of Education Department of Yunnan Province(2013Y251)Characteristic Biological Resource Development and Utilization Key Laboratory Open Fund of Kunming University(GXKJ201612)Fund for Introduction of Doctors(YJL11015)
文摘Abstract Leaves of melon were collected and extracted by the CTAB method for total DNA which was used for PCR amplification, obtaining the gene sequence of cucumisin promoter. The sequence results were processed and analyzed with DNAman, DNAstar and other softwares, and bioinformatic element analysis was performed with PlantCARE and PLACE. The analysis results showed that the cucumisin promoter shared 100%, 99% and 99% homology with AY055805, LN713264 and LN681897, respectively. The promoter sequence contains a variety of c/s-acting elements common in fruit promoters of higher plants such as TATA-Box and CAAT-Box, and light-responsive elements, some of which involved in ABA and VP1 responsiveness and salicylic acid responsiveness. This study provides a scien- tific basis for further research on genetic engineering of fruits.
基金supported by the National Natural Science Foundation of China,No.81671469,81171072(to ZWY)the National Basic Research Program of China(973 Program),No.2013CB945402(to ZWY)the Program for Liaoning Innovative Research Team in University of China,No.LT2013016(to ZWY)
文摘O6-methylguanine DNA methyltransferase(MGMT), a DNA repair enzyme, has been reported in some congenital malformations, but it is less frequently reported in neural tube defects. This study investigated MGMT mRNA expression and methylation levels in the early embryo and in different embryonic stages, as well as the relationship between MGMT and neural tube defects. Spina bifida aperta was induced in rats by a single intragastric administration of all-trans retinoic acid on embryonic day(E) 10, whereas normal control rats received the same amount of olive oil on the same embryonic day. DNA damage was assessed by detecting γ-H2 A.X in spina bifida aperta rats. Real time-polymerase chain reaction was used to examine mRNA expression of MGMT in normal control and spina bifida aperta rats. In normal controls, the MGMT mRNA expression decreased with increasing embryonic days, and was remarkably reduced from E11 to E14, reaching a minimum at E18. In the spina bifida aperta model, γ-H2 A.X protein expression was increased, and mRNA expression of MGMT was markedly decreased on E14, E16, and E18. Bisulfite sequencing polymerase chain reaction for MGMT promoter methylation demonstrated that almost all CpG sites in the MGMT promoter remained unmethylated in both spina bifida aperta rats and normal controls, and there was no significant difference in methylation level between the two groups on either E14 or E18. Our results show that DNA damage occurs in spina bifida aperta rats. The mRNA expression of MGMT is downregulated, and this downregulation is independent of promoter DNA methylation.
基金Supported by the Biomedical Research Councilthe Institute of Bioengineering and Nanotechnology,the Republic of Singapore
文摘AIM: The GFAP was traditionally considered to be a biomarker for neural gila (mainly astrocytes and nonmyelinating Schwann cells). Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astrocytes in vitro and in vivo. More recently, GFAP was also established as one of the several makers for identifying hepatic stellate cells (HSC). In this project, possible application of the same 2.2-kb human GFAP promoter for targeting HSC was investigated. METHODS: The GFAP-lacZ transgene was transfected into various cell lines (HSC, hepatocyte, and other nonHSC cell types). The transgene expression specificity was determined by X-gal staining of the β-galactosidase activity. And the responsiveness of the transgene was tested with a typical pro-fibrotic cytokine TGF-β1. The expression of endogenous GFAP gene was assessed by real-time RT-PCR, providing a reference for the transgene expression. RESULTS: The results demonstrated for the first time that the 2.2 kb hGFAP promoter was not only capable of directing HSC-specific expression, but also responding to a known pro-fibrogenic cytokine TGF-β1 by upregulation in a doseand time-dependent manner, similar to the endogenous GFAP. CONCLUSION: In conclusion, these findings suggested novel utilities for using the GFAP promoter to specifically manipulate HSC for therapeutic purpose.
基金This work was supported by grant from the National Natural Science Foundation of Chinaspecial grant from the Biosystematics Program of Academia Sinica.
文摘The 3.1 kb BamHI fragment containing the chloroplast rbcL gene from grape (Vitis vinifera L) hasbeen cloned and its restriction map and nucleotide sequence determined. The complete nucleotidesequence is 20O4 bp long with a coding sequence of 1428 bp, which cncode a polypeptide of 475amino acids with a predicted molecular weight of 53 kd. The 5' upstream sequence including theputative promoter is 358 bp, with a -10 sequence (TAAAAT), a -35 sequence (TTGCGC) and theSD sequence (GGAGG). The 218 bp long 3' downstream sequence contains three transcription stem-loop tendnation structures. The homologies of this gene with those of tobacco, petunia, spinach,alfalfa, rice and maise are 91.5%, 91.4% 90.2%, 89.8%, 86.3% and 84.5% respectively while thehomologies between their putative polypeptide sequences are 92.2% 91.6%, 92.2%, 93.7%, 93.5%and 90.1% respectively.
文摘A 3.2 kb EcoRI DNA fragment containing the complete chloroplast rbcL gene from Acanthopanax brachypus has been cloned. In addition to the 1428 bp coding region encoding 475 amino acids of the gene, 278 bp upstream and 218 bp downstream were also sequenced. Possible ctpl and ctp2, equivalent to prokaryotic-35 and -10 elements, were found as TTGCGC and TACAAT respectively. The 5' untranslated leader region is 194 bp and the putative ribosome binding site in this region is GGAGG,located immediately upstream of the start codon. The 3' unrtanslated region contains two inverted repeat sequences, which in the mRNA might form stem-loop structures. The homology of rbcL amino acid sequence between A. brachypus and tobacco, spinach, pea, alfalfa, maize, rice, pine,Marchantia, Chlamydomonas and Anacystis are, respectively 93.05%,94.11%, 94.53%,89.68%, 92.21%, 2.21%, 92.63%, 87.58% and 80.84%.The promoter regions and part of the 5', 3' non-coding regions of rbcL from various plants were compared.
基金supported by the national key research and development projects (2017YFD0500500)the national natural science foundation of China(31972529, 31902184)the China postdoctoral science foundation (2019M653774)。
文摘Background: Antibiotic growth promoters(AGPs) have been used as growth promoters to maintain animal intestinal health and improve feed efficiency in broilers by inhibiting pathogen proliferation. In view of the growing emergence of antibiotic-resistant pathogen strains and drug residue issues, novel treatments are increasingly required. This study aimed to compare two antimicrobial approaches for managing pathogen infection and maintaining animal intestinal health in broilers by supplying Apidaecin Api-PR19 and AGPs over 42 d of a feeding trial.Results: Compared with the broilers that were only fed a corn-soybean basal diet(CON group), supplementation with Api-PR19 and AGP(respectively named the ABP and AGP groups) both increased the feed conversion efficiency. When compared with the AGP group, Api-PR19 supplementation could significantly increase the organ index of the bursa of fabricius and subtype H9 antibody level in broiler chickens. Moreover, when compared with the CON group, the intestinal villus height, intestinal nutrient transport, and intestinal s Ig A content were all increased in the Api-PR19 group, while AGP supplementation was harmful to the intestinal villus height and intestinal nutrient transport. By assessing the antibacterial effect of Api-PR19 and antibiotics in vitro and in vivo, we found that Api-PR19 and antibiotics both inhibited the growth of pathogens, including Escherichia coli and Campylobacter jejuni. Furthermore, by using 16 S r RNA gene sequencing, the beneficial bacteria and microbiota in broilers were not disturbed but improved by apidaecin Api-PR19, including the genera of Eubacterium and Christensenella and the species of uncultured_Eubacterium_sp, Clostridium_asparagiforme, and uncultured_Christensenella_sp, which were positively related to improved intestinal development, absorption, and immune function.Conclusion: Apidaecin Api-PR19 treatment could combat pathogen infection and had little negative impact on beneficial bacteria in the gut compared to antibiotic treatment, subsequently improving intestinal development,absorption, and immune function.
文摘The biosafety of genetically engineered plants has been of concernment in society and science in recent years. The issue of 35S promoter of CaMV has been contentious because of its wide use in plant genetic engineering. The debate on the safety and potential risks of the 35S promoter will be discussed here. Some of concerns are expressed about the dissemination of antibiotic_resistance genes and vector backbone sequences. Various methods and strategies are currently being developed for the marker gene excision and elimination of vector backbone sequences from transgenic plants. In this review, the CRE/ lox system which could get rid of the marker geens and vector backbone sequences will be discussed in detail. Advances in the research of the safety assessment of genetically modified plants using the CRE/ lox system will also be described.