Expression of the truncated E. coli O6-methylguanine methyltransferase gene in repair-deficient human cells and restoration of cellular resistance to alkylating agents

We have constructed a truncated E. coli O6-methylguanine methyltransferase (MT) gene (ada gene) to express the MT activity for O6-methylguanine and O4-methylthymine but not for methylphosphotriester in human cells and transferred it into Mer- HeLa MR cells. The transfectant cells expressed the truncated E. coli MT were resistant to alkylating agents as same as the transfectant cells with the intact ada gene in cell killing, sister-chromatid exchange induction and host-cell reactivation of adenovirus 5. These results strongly suggest that methylphosphotriester may not contribute to the biological effect of alkylating agents in human cells.     

麻疹病毒血凝素基因工程抗原及其抗原性检测(英文)

将麻疹病毒 (Nepal株 )的血凝素 (hemagglutinin)基因插入真核表达载体pIRES EGFP ,并在HeLa细胞中表达 .因其较低的表达量 ,所以将其截短 ,去除跨膜区 .使这个截短的HA基因与绿色荧光蛋白基因融合 ,并克隆至原核表达载体pET 2 8b中 .将重组质粒转入大肠杆菌中表达 ,产生了分子量约为 90kD的融合蛋白 .通过ELISA和Western印迹来检测这个基因工程蛋白的抗原性 .在检测一系列的血凝素阳性或阴性的人血清中 ,这个融合蛋白的阳性检出率为 90 % ,阴性检出率为 10 0 % (与市售麻疹病毒诊断试剂盒相比较 ) .由于此HA蛋白是原核表达产物 ,回避了真核表达系统复杂的操作过程和昂贵的费用 ,所以 ,这个麻疹病毒血凝素基因工程抗原有望成为一种新型、便捷的麻疹病毒诊断试剂     

HeLa cell invasion by a strain of enteropathogenic Escherichia coli that lacks the O-antigenic polysaccharide

The interaction with HeLa cells of an enteropathogenic Escherichia coli (EPEC) strain and its plasmid-cured derivative strain was examined. An O111:NM EPEC strain B171 harbours a 54 megadalton plasmid (pYR111) necessary for the expression of both localized adherence (LA) to HeLa cells and the O-repeating side chain of the lipopolysaccharide. Under light microscopy, the plasmid-cured derivative strain B171-4 was observed to interact with HeLa cells in a pattern distinct from LA. Transmission electron microscopy showed that the bacteria were internalized by HeLa cells. In contrast, strain B171 induced pedestal-like projections and invaginations of the plasma membrane, but was never completely internalized. A quantitative assay to determine the number of internalized bacteria revealed that strain B171-4 was internalized at levels 30-70-fold higher than those of avirulent E. coli strains. Cytochalasin B reduced the levels of internalization of both strain B171-4 and an enteroinvasive E. coli strain (E11), but did not affect LA by strain B171. These results suggest that EPEC strain B171 may carry a specific chromosomally determined surface factor needed to initiate internalization by HeLa cells. However, a plasmid-determined factor alters the nature of this interaction; the combined effects of the chromosomal and plasmid determinants lead to the characteristic attachment of the bacteria in clusters on the surface of the eukaryotic cell.     

Construction of a fusion gene that confers resistance against hygromycin B to mammalian cells in culture

Mouse L fibroblasts and other mammalian cells are killed by the translation inhibitor hygromycin B. We have modified the gene conferring resistance against hygromycin B in E. coli in such a way that it can be transcribed in mammalian cells from the promoter of the HSVtk gene. The resulting plasmid, pHMR272, was transfected into mouse L fibroblasts and HeLa cells by the calcium phosphate method and upon selection produced clones resistant against hygromycin B. The transfection rate was similar to that obtained with other selective markers. This plasmid is a useful addition to the relatively small number of dominant selectable markers available for mammalian cells.     

Expression of the chloramphenicol acetyl transferase gene in human cells under the control of early adenovirus subgroup C promoters: effect of E1A gene products from other subgroups on gene expression

A hierarchy of dominance has been observed in HeLa cells co-infected with two serotypes of adenovirus belonging to different subgroups. DNA replication and late protein synthesis of one serotype are inhibited by those of the other. The degree of inhibitory effect has the following decreasing order: adenovirus type 3 (Ad3) and Ad7 (subgroup B), Ad9 (D), Ad4 (E), Ad12 (A), Ad2 and Ad5 (C) [Delsert and D'Halluin, Virus Res. 1 (1984) 365-380]. HeLa cells were first transfected with recombinant plasmids carrying Ad5 E2A or E3 promoters fused to the chloramphenicol acetyl transferase gene (cat), and then infected with human Ad belonging to different subgroups. All the serotypes tested were found to be able to stimulate both E2A and E3 promoters. When HeLa cells were co-transfected with either of the previous plasmids, plus a second plasmid carrying the Ad3 E1A region, the same stimulatory effect was observed. However, an inhibitory effect on Ad5 E2A and E3 promoters seemed to occur when both Ad2 E1A (subgroup C) and Ad3 E1A (subgroup B) genes were present together. To determine which one of the early products was responsible for the observed repression effect, and to assign the target on the genome of subgroup C Ad, a plasmid was constructed in which the sequences at the 5' end of the Ad2 E1A region were fused to the structural sequences of the cat gene. In HeLa cells transfected with this plasmid, CAT activity was significantly increased after co-transfection with a plasmid carrying the Ad2 E1A region, but decreased with a plasmid carrying the Ad3 E1A region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sister-chromatid exchanges (SCEs), cell survival and mutation in HeLa s3 cells with different sensitivity to alkylating agents; evidence that SCE induction and cell survival or mutation induction are dissociable

We previously isolated N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-resistant cells, MR from HeLa S3 Mer- cells. In the present study, we have isolated 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU)-resistant cells, ACr. The MR cells had only a little O6-methylguanine-DNA methyltransferase (MT) activity, while the ACr cells had increased MT activity and also became resistant to the cytotoxic effect of MNNG. We compared the induction of sister-chromatid exchanges (SCEs), cell survival and mutation in these HeLa S3 cells with different sensitivity to MNNG. The ACr cells were much more resistant than the parental HeLa S3 Mer- cells to cytotoxicity, mutagenicity and SCE induction by MNNG, showing a positive correlation between SCE induction and cell killing or mutation. In contrast, this positive relationship was not observed between HeLa S3 Mer- and MR cells. These results suggest that O6-methylguanine (O6-MeG) is involved in the induction of the biological effects of MNNG such as cytotoxicity, mutagenicity and SCEs, and also indicate that SCE induction does not always correlate with cell killing and mutation.     

Bioluminescent high-throughput assay for the bacteria adherence to the tissue culture cells

The goal of this study was develop a rapid high-throughput method for the assessment of the bacterial adhesion to tissue culture cells and test this method by investigation of the adhesion and growth of pathogenic and non-pathogenic Escherichia coli strains in the presence of HeLa human epithelial cells. Fifteen strains of E. coli were transformed with a plasmid carrying the entire lux operon of Photorhabdus luminescens to make them bioluminescent. By using the Time-to-Detection approach and bioluminescence imaging in microplate format, the adherence and growth of bacteria in tissue culture medium in the presence of HeLa cells was monitored. It was observed that Eagle's minimal essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS) significantly inhibited growth of E. coli. However, in the presence of HeLa cells the detected growth of E. coli was similar to the growth observed in LB medium. It was established that the initial number of E. coli cells present in the microplate directly correlated with the time necessary for the bioluminescence signal to reach the threshold level, hence allowing the accurate assessment of the adhered cells within 8-10 h. Neither bacterial adherence nor growth kinetics correlated with the pathogenicity of the strain though they were strain-specific. The developed approach provided new information on the interaction of E. coli with epithelial cells and could be used for both pathogenicity research and for the screening of potential therapeutic agents for the ability to minimize pathogen colonization of human tissues.     

Development of a new shuttle plasmid system for Escherichia coli and Clostridium perfringens.

We constructed a 7.9-kilobase-pair recombinant shuttle plasmid, designated pHR106, by combining desired segments of three plasmids: an Escherichia coli plasmid (pSL100) which provides a multiple cloning site, a Clostridium perfringens plasmid (pJU122) which provides a clostridial origin of replication, and an E. coli plasmid (pJIR62) which provides an E. coli origin of replication, an ampicillin resistance gene, and a chloramphenicol resistance gene of clostridial origin. The shuttle plasmid transformed E. coli HB101 with a frequency of 1 transformant per 10(4) viable cells and C. perfringens L-phase strain L-13 with a frequency of approximately 1 transformant per 10(6) viable cells. Because of the set of unique cloning sites and the chloramphenicol resistance marker, this shuttle plasmid should be particularly useful for studies of gene regulation and for enzyme production with C. perfringens.     

Enhanced expression of a recombinant malaria candidate vaccine in Escherichia coli by codon optimization

This study was conducted to compare the expression of three constructs of a multistage candidate vaccine (FALVAC-1) against Plasmodium falciparum in an Escherichia coli system: a synthetic gene with P. falciparum codons, a synthetic gene with optimized E. coli codons, and a synthetic gene with P. falciparum codons co-transformed with a RIG plasmid, which encodes three tRNAs (AG(A/G), ATA, GGA) that recognize rare E. coli codons. The expression of the protein increased at least threefold with codon optimization. The presence of the RIG plasmid in the co-transforming cells did not significantly increase the expression level of the gene with P. falciparum codons. The growth of cells transformed by the construct with P. falciparum codons was significantly slower than that of cells transformed by the construct with optimized E. coli codons after induction of protein expression with IPTG. The cells containing the non-codon optimized gene co-expressed with RIG plasmid had the slowest growth at all time points in culture. Thus, codon optimization significantly increases the yield of P. falciparum candidate vaccines in the E. coli expression system.     

Cloning of the E. coli O6-methylguanine and methylphosphotriester methyltransferase gene using a functional DNA repair assay.

Alkylating agents react with various nitrogen and oxygen atoms in DNA and many of the products are substrates for repair processes. Oxygen atom derivatives such as O6-methylguanine (O6-meG) O4-methylthymine and methylphosphotriesters (MP) have been shown to undergo repair by methyl group removal. The proteins involved in the latter reaction can be considered to be methyltransferases (MT) because their action results in the transfer of the methyl group to a cysteine residue within a polypeptide. A rapid and sensitive assay for MT activity has been developed and used to screen extracts of bacteria harbouring an E. coli genomic DNA library carried in a plasmid vector. We report here the cloning of an E. coli gene coding for O6-meG and MP MT repair functions. These two activities reside on a 37Kd protein that can undergo a host-dependent cleavage to produce an 18Kd protein which contains only O6-meG MT and a 13Kd protein which contains only MP MT.     

Acquisition of a sucrose utilization system in Escherichia coli K-12 derivatives and its application to industry.

An Escherichia coli strain, B-62, that was isolated from a clinical source and was epidemiologically unrelated to E. coli K-12 was the source of chromosomal DNA for a sucrose utilization system (Scr+) in the construction of a plasmid, pST621. The cloned insert of a gene encoding Scr+ in pST621 conferred a sucrose-positive phenotype onto transformed cells of E. coli K-12 derivatives. Sucrase activity of the transformants was as high as that which would correspond to a "gene dosage effect" of a vector plasmid pBR322, whereas the transformants' sucrose uptake activity was always lower than that of E. coli B-62. A region within an XhoI-SacI fragment (3.2 kb) of pBR322-glyA was replaced in the construction of another plasmid, pST5R7, by a fragment (about 2.6 kb) of pST622 containing the gene encoding Scr+. A genetically stable Scr+ derivative of E. coli K-12 was obtained by introducing the gene encoding Scr+ onto E. coli chromosome via homologous recombination between pST5R7 and the chromosome and subsequent plasmid segregation. The use of low-copy-number plasmid RP4 as a cloning vector was also effective for enhancing the stability of Scr+. Tryptophan producers E. coli SGIII1032S, in which the gene encoding Scr+ was cloned onto the chromosome, and E. coli SGIII1032, which carried Scr+ plasmid RP4.5R7, produced from 6% sucrose in shake flasks (33 degrees C, 96 h) 2.3 and 5.7 g of tryptophan per liter, respectively.     

Acquisition of a sucrose utilization system in Escherichia coli K-12 derivatives and its application to industry.

An Escherichia coli strain, B-62, that was isolated from a clinical source and was epidemiologically unrelated to E. coli K-12 was the source of chromosomal DNA for a sucrose utilization system (Scr+) in the construction of a plasmid, pST621. The cloned insert of a gene encoding Scr+ in pST621 conferred a sucrose-positive phenotype onto transformed cells of E. coli K-12 derivatives. Sucrase activity of the transformants was as high as that which would correspond to a "gene dosage effect" of a vector plasmid pBR322, whereas the transformants' sucrose uptake activity was always lower than that of E. coli B-62. A region within an XhoI-SacI fragment (3.2 kb) of pBR322-glyA was replaced in the construction of another plasmid, pST5R7, by a fragment (about 2.6 kb) of pST622 containing the gene encoding Scr+. A genetically stable Scr+ derivative of E. coli K-12 was obtained by introducing the gene encoding Scr+ onto E. coli chromosome via homologous recombination between pST5R7 and the chromosome and subsequent plasmid segregation. The use of low-copy-number plasmid RP4 as a cloning vector was also effective for enhancing the stability of Scr+. Tryptophan producers E. coli SGIII1032S, in which the gene encoding Scr+ was cloned onto the chromosome, and E. coli SGIII1032, which carried Scr+ plasmid RP4.5R7, produced from 6% sucrose in shake flasks (33 degrees C, 96 h) 2.3 and 5.7 g of tryptophan per liter, respectively.     

Uracil-DNA glycosylase inhibitor of bacteriophage PBS2: cloning and effects of expression of the inhibitor gene in Escherichia coli.

The uracil-DNA glycosylase inhibitor gene of bacteriophage PBS2 was cloned, and the effects of this inhibitor on Escherichia coli cells that contain uracil-DNA glycosylase activity were determined. A PBS2 genomic library was constructed by inserting EcoRI restriction fragments of PBS2 DNA into a plasmid pUC19 vector. The library was used to transform wild-type (ung+) E. coli, and the presence of the functional inhibitor gene was determined by screening for colonies that supported growth of M13mp19 phage containing uracil-DNA. A clone was identified that carried a 4.1-kilobase EcoRI DNA insert in the vector plasmid. Extracts of cells transformed with this recombinant plasmid lacked detectable uracil-DNA glycosylase activity and contained a protein that inhibited the activity of purified E. coli uracil-DNA glycosylase in vitro. The uracil-DNA glycosylase inhibitor expressed in these E. coli was partially purified and characterized as a heat-stable protein with a native molecular weight of about 18,000. Hence, we conclude that the PBS2 uracil-DNA glycosylase inhibitor gene was cloned and that the gene product has properties similar to those from PBS2-infected Bacillus subtilis cells. Inhibitor gene expression in E. coli resulted in (i) a weak mutator phenotype, (ii) a growth rate similar to that of E. coli containing pUC19 alone, (iii) a sensitivity to the antifolate drug aminopterin similar to that of cells lacking the inhibitor gene, and (iv) an increased resistance to the lethal effects of 5-fluoro-2'-deoxyuridine. These physiological properties are consistent with the phenotypes of other ung mutants.     

Cloning of a gene from Pseudomonas sp. strain PG2982 conferring increased glyphosate resistance

A plasmid carrying a 2.4-kilobase-pair fragment of DNA from Pseudomonas sp. strain PG2982 has been isolated which was able to increase the glyphosate resistance of Escherichia coli cells. The increase in resistance was dependent on the presence of a plasmid-encoded protein with a molecular weight of approximately 33,000, the product of a translational fusion between a gene on the vector, pACYC184, and the insert DNA. An overlapping region of the PG2982 chromosome carrying the entire gene (designated igrA) was cloned, and a plasmid (pPG18) carrying the gene was also able to increase glyphosate resistance in E. coli. A protein with a molecular weight of approximately 40,000 was encoded by the PG2982 DNA contained in pPG18. This plasmid was not able to complement a mutation in the gene for 5-enolpyruvylshikimate-3-phosphate synthase (aroA) in E. coli, and modification of glyphosate by E. coli cells containing the plasmid could not be demonstrated. The nucleotide sequence of the PG2982 DNA contained an open reading frame able to encode a protein with a calculated molecular weight of 39,396.     

Inactive O6-methylguanine-DNA methyltransferase in human cells.

A plasmid encoding a recombinant human O6-methylguanine-DNA methyltransferase (MGMT) fused to a fragment of the bacteriophage lambda N protein has been constructed. The fusion protein retained methyltransferase activity when expressed at high levels in E.coli and was purified to essential homogeneity by a simple procedure. Antisera raised against the purified fusion protein recognized MGMT in western blots of extracts of human cells. For most cell lines, there was a quantitative relation between the amount of immunologically detectable MGMT protein and enzyme activity. However, four cell lines contained detectable MGMT protein despite having no measurable methyltransferase activity. Additionally, a HeLa line contained considerably more immunoreactive MGMT protein than could be accounted for by its methyltransferase activity. Thus, some cells contain significant amounts of inactive MGMT. Preliminary characterization of the inactive protein in HeLaS3 cells indicated that it has some properties in common with MGMT methylated at the active cysteine residue.     

Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli

Real-time QPCR based methods for determination of plasmid copy number in recombinant Escherichia coli cultures are presented. Two compatible methods based on absolute and relative analyses were tested with recombinant E. coli DH5alpha harboring pBR322, which is a common bacterial cloning vector. The separate detection of the plasmid and the host chromosomal DNA was achieved using two separate primer sets, specific for the plasmid beta-lactamase gene (bla) and for the chromosomal d-1-deoxyxylulose 5-phosphate synthase gene (dxs), respectively. Since both bla and dxs are single-copy genes of pBR322 and E. coli chromosomal DNA, respectively, the plasmid copy number can be determined as the copy ratio of bla to dxs. These methods were successfully applied to determine the plasmid copy number of pBR322 of E. coli host cells. The results of the absolute and relative analyses were identical and highly reproducible with coefficient of variation (CV) values of 2.8-3.9% and 4.7-5.4%, respectively. The results corresponded to the previously reported values of pBR322 copy number within E. coli host cells, 15-20. The methods introduced in this study are convenient to perform and cost-effective compared to the traditionally used Southern blot method. The primer sets designed in this study can be used to determine plasmid copy number of any recombinant E. coli with a plasmid vector having bla gene.     

Cloning of a gene from Pseudomonas sp. strain PG2982 conferring increased glyphosate resistance.

A plasmid carrying a 2.4-kilobase-pair fragment of DNA from Pseudomonas sp. strain PG2982 has been isolated which was able to increase the glyphosate resistance of Escherichia coli cells. The increase in resistance was dependent on the presence of a plasmid-encoded protein with a molecular weight of approximately 33,000, the product of a translational fusion between a gene on the vector, pACYC184, and the insert DNA. An overlapping region of the PG2982 chromosome carrying the entire gene (designated igrA) was cloned, and a plasmid (pPG18) carrying the gene was also able to increase glyphosate resistance in E. coli. A protein with a molecular weight of approximately 40,000 was encoded by the PG2982 DNA contained in pPG18. This plasmid was not able to complement a mutation in the gene for 5-enolpyruvylshikimate-3-phosphate synthase (aroA) in E. coli, and modification of glyphosate by E. coli cells containing the plasmid could not be demonstrated. The nucleotide sequence of the PG2982 DNA contained an open reading frame able to encode a protein with a calculated molecular weight of 39,396.     

Generation of various amino acids mutants in the trpR gene of Escherichia coli by site-directed mutagenesis

Tryptophan repressor (trpR) gene lacks various amino acid codons. To establish these codons in the trpR gene, we created the mutants by site-directed mutagenesis in the trpR gene of pHK1 plasmid. The interested regions of trpR gene were amplified, cloned in pT7-5 plasmid and transformed in to the cells harboring pGP1-2 plasmid. These plasmid products were labeled with (35)S Met, and following sequencing we observed the presence of mutants for cysteine, glycine, serine and lysine in the trpR gene of E. coli. Therefore, using these approach mutants in various genes of E. coli could be established and used as a tool to study translational bypassing in trpR gene of E. coli.     

Transfer of the Ti plasmid from Agrobacterium tumefaciens into Escherichia coli cells

We have screened strains of Agrobacterium tumefaciens for spontaneous mutants showing constitutive transfer of the nopaline Ti plasmid pTiC58 during conjugation. The Ti plasmid derivatives obtained could be transferred not only to A. tumefaciens but also to E. coli cells. The Ti plasmid cannot survive as a freely replicating plasmid in E. coli, but it can occasionally integrate into the E. coli chromosome. However, insertion in tandem of plasmids carrying fd replication origins (pfd plasmids) into the T-DNA provides an indicator for all transfer events into E. coli cells, providing fd gene 2 protein is present in these cells. This viral protein causes the excision of one copy of the pfd plasmid and allows its propagation in the host cell. By using this specially designed Ti plasmid, which was also made constitutive in transfer functions, we found plasmid exchange among A. tumefaciens strains and between A. tumefaciens and E. coli cells to be equally efficient. A Ti plasmid with repressed transfer functions was transferred to E. coli with a rate similar to the low frequency at which it was transferred to A. tumefaciens. The expression of transfer functions of plasmid RP4 either in A. tumefaciens or in E. coli did not increase the transfer of the Ti plasmid into E. coli cells, nor did the addition of acetosyringone, an inducer of T-DNA transfer to plant cells. The results show that A. tumefaciens can transfer the Ti plasmid to E. coli with the same efficiency as within its own species. Conjugational transmission of extrachromosomal DNA like the narrow-host-range Ti plasmid may often not only occur among partners allowing propagation of the plasmid, but also on a 'try-all' basis including hosts which do not replicate the transferred DNA.     

Molecular cloning of the Pseudomonas putida glyoxalase I gene in Escherichia coli

The glyoxalase I gene of Pseudomonas putida was cloned onto a vector plasmid pBR 322 as a 7.5 kilobase Sau 3AI fragment of chromosomal DNA and the hybrid plasmid was designated pGI 318. The gene responsible for the glyoxalase I activity in pGI 318 was recloned in pBR 322 as a 2.2 kilobase Hin dIII fragment and was designated pGI 423. The P. putida glyoxalase I gene on pGI 318 and pGI 423 was highly expressed in E. coli cells and the glyoxalase I activity level was increased more than 150 fold in the pGI 423 bearing strain compared with that of E. coli cells without pGI 423. The E. coli transformants harboring pGI 318 or pGI 423 could grow normally in the presence of methylglyoxal, although the E. coli cells without plasmid were inhibited to grow and showed the extremely elongated cell shape.