Analyses of mutation in pSV2gpt-transformed CHO cells

We have developed a system to study mutations which affect expression of the E. coli xanthine-guanine phosphoribosyl transferase (XPRT) gene (gpt) in hypoxanthine-guanine phosphoribosyl transferase-deficient (HPRT-) Chinese hamster ovary (CHO) cells that have been transformed by the plasmid pSV2gpt. Several gpt-transformed cell lines have been isolated and characterized with respect to integrated pSV2gpt sequences, expression of the gpt gene, and cytotoxic and mutagenic responses to UV light. While the gpt-transformed CHO and wild-type CHO-K1-BH4 cell lines have similar cytotoxic responses to UV light, the gpt-transformed cell lines respond differently from the parental CHO-K1-BH4 cell line in terms of mutation induction. As with CHO-K1-BH4 HPRT mutants, spontaneous or induced XPRT mutants derived from the gpt+ cell lines can be selected for 6-thioguanine resistance (TGr). Analysis of cell-free extracts from a number of these TGr clones indicates that the mutant phenotype is due to the absence of XPRT activity. One transformant, designated AS52, has previously been described in limited detail. Here we describe additional characteristics of this cell line, as well as several related transformants.     

Transfer of the E. coli O6-methylguanine methyltransferase gene into repair-deficient human cells and restoration of cellular resistance to N-methyl-N'-nitro-N-nitrosoguanidine

We have constructed a plasmid on which the E. coli O6-methylguanine-DNA methyltransferase (MT) gene (ada gene) was linked with an SV40 promoter sequence and a poly(A) site. After transferring this plasmid into Mer- HeLa MR cells by DNA transfection, effective expression of E. coli MT was observed. Isolated stable transformant clones showed higher resistance to N-methyl-N'-nitro-N-nitrosoguanidine in colony formation and sister-chromatid exchange induction than HeLa MR cells.     

Cloning the complete human adenine phosphoribosyl transferase gene

We have isolated a clone from a human genomic lambda library which cross-hybridises with the cloned hamster adenine phosphoribosyl transferase gene (aprt). After restriction mapping and further hybridisation to the hamster gene, a series of putative human aprt-containing fragments has been isolated and tested for ability to transform adenine phosphoribosyl transferase-deficient (aprt-) strains of Chinese hamster ovary (CHO) cells to APRT proficiency. Transforming activity was detected in a 48-kb lambda clone, the 17.4-kb EcoRI insert, and an 8.6-kb HincII fragment. Smaller fragments have thus far shown no transforming activity. Transformants appear to be stable for the APRT+ phenotype, and human aprt DNA sequences are present in the hamster transformants. The 8.6-kb HincII fragment has been subcloned and the insert mapped. Nonrepetitive regions of this subclone have been identified, and should prove valuable for chromosome walking studies on human chromosome 16, familial studies of a human aprt- trait, the analysis of restriction fragment length polymorphisms (RFLPs) in the area surrounding the aprt gene, and the fine structure mapping of the mutations induced by chemical carcinogens and alkylating agents.     

Transformation of UV-hypersensitive Chinese hamster ovary cell mutants with UV-irradiated plasmids

Transfection of UV-hypersensitive, DNA repair-deficient Chinese hamster ovary (CHO) cell lines and parental, repair-proficient CHO cells with UV-irradiated pHaprt-1 or pSV2gpt plasmids resulted in different responses by recipient cell lines to UV damage in transfected DNA. Unlike results that have been reported for human cells, UV irradiation of transfecting DNA did not stimulate the genetic transformation of CHO recipient cells. In repair-deficient CHO cells, proportionally fewer transformants were produced with increasing UV damage than in repair-proficient cells in transfections with the UV-irradiated hamster adenine phosphoribosyltransferase (APRT) gene contained in plasmid pHaprt-1. However, transfection of CHO cells with UV-irradiated pSV2gpt resulted in neither decline in transformation frequencies in repair-deficient cell lines relative to repair-proficient cells nor stimulation of genetic transformation by UV damage in the plasmid. Blot hybridization analysis of DNA samples isolated from transformed cells showed no dramatic changes in copy number or arrangement of transfected plasmid DNA with increasing UV dose. We conclude that the responses of recipient cells to UV-damaged transfecting plasmids depend both on the type of recipient cell and the characteristics of the genetic sequence used for transfection.     

Cloning and expression of a mouse adenine phosphoribosyltransferase gene

A functional mouse adenine phosphoribosyltransferase (APRT) gene was identified and cloned by screening a mouse sperm genomic DNA library in lambda Charon 4A. The probe utilized for screening was a restriction fragment encoding much of the hamster APRT gene. Six recombinants that hybridized with the probe were identified, and after digestion with restriction enzymes EcoRI and PvuII revealed three different patterns of digestion for each enzyme. Of the six recombinants, five representing two of the restriction patterns possessed transforming activity. A sixth recombinant, which has a unique restriction pattern, lacks transforming activity but hybridizes well with hamster APRT coding sequences and is a possible candidate for a pseudogene. We used three criteria for conclusively identifying the mouse APRT genes. (1) DNA from the recombinant lambda phage hybridizes with DNA encoding hamster APRT. (2) The recombinant lambda phages and their DNAs transform mouse, hamster and human APRT- cells to the APRT+ phenotype. (3) The hamster and human transformants display APRT activity that migrates with a mobility characteristic of mouse APRT and not of hamster or human. A 3.1-kb EcoRI-SphI restriction fragment which retains transforming activity has been subcloned into the plasmid pBR328. Comparison of restriction enzyme sites with those contained in a mouse APRT cDNA, coupled with loss of transforming activity after enzyme digestion, indicates that the mouse APRT gene is larger than 1.8 kb and contains at least three introns.     

Expression of Ca(2+)-induced Ca2+ release channel activity from cardiac ryanodine receptor cDNA in Chinese hamster ovary cells.

We constructed an expression plasmid (pMAMCRR51) that carried the entire protein-coding sequence of the rabbit cardiac ryanodine receptor cDNA, linked to the dexamethasone-inducible mouse mammary tumor virus promoter and Escherichia coli xanthine-guanine phosphoribosyltransferase (gpt). Chinese hamster ovary (CHO) cells were transfected with pMAMCRR51 and mycophenolic acid-resistant cells showing caffeine-induced intracellular Ca2+ transients were selected. Immunoprecipitation with a monoclonal antibody against the canine cardiac ryanodine receptor revealed that the cell clones thus selected exhibited Ca(2+)-dependent [3H]ryanodine binding activity, which was stimulated by 5 mM ATP or 1 M KCl. The apparent dissociation constant (Kd) for [3H]ryanodine was 6.6 nM in 1 M KCl, which was similar to the Kd obtained with cardiac microsomes. Immunoprecipitation also demonstrated that these cell clones expressed a protein indistinguishable in M(r) from the ryanodine receptor in canine cardiac microsomes. The ryanodine binding activity expressed in CHO cells increased significantly after dexamethasone induction. In saponin-skinned CHO cells transfected with pMAMCRR51, micromolar Ca2+ or millimolar caffeine evoked rapid Ca2+ release from the intracellular Ca2+ stores. In skinned control CHO cells, we did not observe such Ca2+ release activity. These results clearly demonstrate that the cardiac ryanodine receptor is stably expressed in internal membranes of CHO cells and functions as Ca(2+)-induced Ca2+ release channels.     

Efficient expression of foreign genes in CHO DHFR cellsby electroporation

DHFR-deficient Chinese hamster ovary (CHO DHFR⁻) cells are the most popular mammalian expression system for inducible amplification of transgene. In order to obtain more stable transfected CHO DHFR⁻ cell clones, transfection efficiency of electroporation under different conditions were systemically investigated using plasmid pSV-β-Gal as reporter gene. Transfection efficiency was proportionally increased with pulse duration and number of pulse applied. In addition, higher transfection efficiency was found in high salt extracellular solution (Berg's and Hank's buffers) than in intracellular solution (cytomix buffer) under the same electroporation condition. The highest transfection efficiency in examined conditions was about 1 in 350 cells (or 0.289%) when cells were electroporated with twice pulses at 400 V, 375 μF. The present study offers an optimized guideline for introducing exogenous DNA into CHO DHFR⁻ cells by electroporation.     

Molecular cloning and biological characterization of a human gene, ERCC2, that corrects the nucleotide excision repair defect in CHO UV5 cells.

The UV-sensitive Chinese hamster ovary (CHO) cell line UV5, which is defective in the incision step of nucleotide excision repair, was used to identify and clone a complementing human gene, ERCC2, and to study the repair process. Genomic DNA from a human-hamster hybrid cell line was sheared and cotransferred with pSV2gpt plasmid DNA into UV5 cells to obtain five primary transformants. Transfer of sheared DNA from one primary transformant resulted in a secondary transformant expressing both gpt and ERCC2. The human repair gene was identified with a probe for Alu-family repetitive sequences. For most primary, secondary, and cosmid transformants, survival after UV exposure showed a return to wild-type levels of resistance. The levels of UV-induced mutation at the aprt locus for secondary and cosmid transformants varied from 50 to 130% of the wild-type level. Measurements of the initial rate of UV-induced strand incision by alkaline elution indicated that, whereas the UV5 rate was 3% of the wild-type level, rates of cosmid-transformed lines were similar to that of the wild type, and the secondary transformant rate was about 165% of the wild-type rate. Analysis of overlapping cosmids determined that ERCC2 is between 15.5 and 20 kilobases and identified a closely linked gpt gene. Cosmids were obtained with functional copies of both ERCC2 and gpt. ERCC2 corrects only the first of the five CHO complementation groups of incision-defective mutants.     

Correction of a deletion mutant by gene targeting with an adenovirus vector.

The usefulness of adenovirus type 5 as a vector for homologous recombination was examined in CHO cells by using the adenine phosphoribosyltransferase (aprt) gene. Infection of a hemizygous CHO APRT- cell line containing a 3-bp deletion in exon 5 of the aprt gene with a recombinant adenovirus containing the wild-type gene resulted in restoration of the APRT+ phenotype at a frequency of 10(-5) to 10(-6) per infected cell. A relatively high frequency (approximately 6 to 20%) of the transductants appears to result from a homologous recombination event. The mutation on the chromosomal aprt gene is corrected in the homologous recombinants, and APRT expression is restored to a normal hemizygous level. Neither adenovirus nor exogenous promoter sequences are detected in the homologous recombinants. The remaining transductants result from random integration of the aprt gene with the adenovirus sequence. A number of adenovirus vectors containing different promoter sequences linked to the hamster aprt gene were constructed. A possible role for the promoter region in the homologous recombination event was indicated by the lack of homologous recombination in constructs lacking an active promoter.     

Genetical and Molecular Analyses of qa-2 Transformants in NEUROSPORA CRASSA

Neurospora crassa qa-2+ transformants from five different donor DNA clones were analyzed by genetical and molecular techniques. None of the 32 transformants have the qa-2+ DNA replacing the qa-2- gene in linkage group VII. In one transformant, the qa-2+ DNA was inserted adjacent to the qa-2- gene. Thirty-one transformants have the qa-2+ inserts at sites not linked, or not closely linked, to the qa-2 locus in LG VII. Plasmid sequences were integrated along with the qa-2+ gene in 28 transformants. In the unlinked duplication-type transformants, catabolic dehydroquinase (the qa-2+ gene product) was induced at 5-100% of the wild-type-induced enzyme activity, with 24 transformants in the 5-80% range. The reduced levels of enzyme activity may be due to "position effects" of sequences adjacent to the integration site either in the N. crassa genomic DNA or in the flanking plasmid (pBR322 or pBR325) sequences. Unexpected gene conversion-like events, in which a qa-2+ gene was changed to qa-2-, were observed in tetrads from intercrosses between unlinked duplication-type transformants and in selfings of such transformants.     

Transfer of genes to Chinese hamster ovary cells by DNA-mediated transformation

We have transferred DNa to Chinese hamster ovary (CHO) cells by DNA-mediated transformation. CHO tk- cells were transformed with the clones gene for herpes simplex virus thymidine kinase (HSV-tk) and were found to have a 50-fold lower frequency of transformation than mouse Ltk- cells at the same DNA dosage. By altering the amount of tk gene and carrier DNA present, frequencies of up to 5 x 10(-5) were obtained. CHO HSV-tk+ transformants were very stable, and in several clones the HSV-tk gene copies integrated in higher-molecular-weight DNA. These cells also exhibited cotransformation for unselected markers. CHO lines were also transformed at a frequency of 10(-4) with the bacterial gene Ecogpt in a SV40-pBR322 vector. CHO tk-cells could be transformed at a frequency of 10(-7) with cellular DNA isolated from CHO tk+ cells. CHO cells offer a well-defined genetic system within which to transfer either cloned or whole cellular DNAs.     

以报告基因分析霍乱弧菌噬菌体VP1的预测启动子

VP1为感染并裂解霍乱弧菌的噬菌体,全基因组为环状双链DNA。通过测定该基因组序列,预测出15个可能的启动子区,利用报告基因质粒转化及全噬菌体共感染的策略分析了这些推测启动子在霍乱弧菌中的活性,将预测的启动子区分别克隆到启动子探测lacZ融合质粒载体pRS1274,在转化于大肠埃希氏菌受体菌株JM109中时,所有克隆子均呈现兰斑。同时将质粒电击到缺失了lacZ基因的霍乱弧菌菌株7743△Z,然后用噬菌体VP1感染转化菌株。在转化成功的13个含预测启动子片段的重组质粒中,通过检测β_半乳糖苷酶活性表达随感染后时间的变化,提示P17为早期启动子,P2、P3、P9等为中期启动子,P18为晚期启动子。     

Cloning and expression of the chromosomal immune interferon gene of the rat.

The chromosomal immune interferon gene of the rat (IFN-gamma) was identified by screening a recombinant rat lambda phage library with a human IFN-gamma cDNA probe. In contrast to the genes of other rat IFNs, this rat IFN-gamma chromosomal gene contains introns and its structural organization closely resembles that of the human and murine IFN-gamma genes. The rat IFN-gamma gene encodes a signal sequence of 19 amino acids followed by the mature IFN-gamma protein of 137 amino acids. The gene was expressed under control of the simian virus 40 (SV40) early promoter in Chinese hamster ovary (CHO) cells deficient in dihydrofolate reductase (DHFR) after co-transformation with a plasmid containing the mouse DHFR gene. Initial transformants with a DHFR+ phenotype produced IFN-gamma titres ranging from 20 to 1600 units/ml. After stepwise increases in the concentration of methotrexate (MTX) in the growth medium of transformed CHO cells, MTX-resistant clones producing 80 000-100 000 units per ml were isolated. Protein analysis of supernatants of these MTX-resistant cells by polyacrylamide gel electrophoresis revealed a product with an apparent mol. wt. of 18 000 daltons which was not detectable in the growth medium of DHFR+ transformants that did not produce IFN. The product was identified as rat IFN-gamma and constituted approximately 5% of the proteins excreted from these cells.     

The origin of O6-methylguanine-DNA methyltransferase in Chinese hamster ovary cells transfected with human DNA

Transfection of Chinese hamster ovary (CHO) cells with human DNA has been shown in several laboratories to produce clones which stably express the DNA-repair protein, O6-methylguanine-DNA methyltransferase (MGMT), that is lacking in the parent cell lines (Mex- phenotype). We have investigated the genetic origin of the MGMT in a number of such MGMT-positive (Mex+) clones by using human MGMT cDNA and anti-human MGMT antibodies as probes. None of the five independently isolated Mex+ lines has human MGMT gene sequences. Immunoblot analysis confirmed the absence of the human protein in the extracts of these cells. The MGMT mRNA in the lines that express low levels of MGMT (0.6-1.4 x 10(4) molecules/cell) is of the same size (1.1 kb) as that present in hamster liver. One cell line, GC-1, with a much higher level of MGMT (4 x 10(4) molecules/cell) has two MGMT mRNAs, a major species of 1.3 kb and a minor species of 1.8 kb. It has also two MGMT polypeptides (32 and 28 kDa), both of which are larger than the 25 kDa MGMT present in hamster liver and other Mex+ transfectants. These results indicate that the MGMT in all Mex+ CHO cell clones is encoded by the endogenous gene. While spontaneous activation of the MGMT gene cannot be ruled out in the Mex+ cell clones, the intervention of human DNA sequences may be responsible for activation of the endogenous gene in the GC-1 line.     

pT7MT, a metallothionein 2A-tagged novel prokaryotic fusion expression vector

In the present article, a novel fusion expression vector for Escherichia coli was developed based on the pTORG plasmid, a derivative of pET32a. This vector, named pT7MT (GenBank Accession No DQ504436), carries a T7 promoter and it drives the downstream gene encoding Metallothionein 2A (MT2A). There are in-framed multiple cloning sites (MCS) downstream of the MT2A gene. A target gene can be cloned into the MCS and fused to the C-terminal of the MT2A gene in a compatible open reading frame (ORF) to achieve fusion expression. The metal-binding capability of MT2A allows the purification of fusion proteins by metal chelating affinity chromatogralhy, known as Ni2+-affinity chromatography. Using this expression vector, we successfully got the stable and high-yield expression of MT2A-GST and MT2A-Troponin I fusion proteins. These two proteins were easily purified from the supernatant of cell lysates by one-step Ni2+ -affinity chromatography. The final yields of MT2A-GST and MT2A-Troponin I were 30 mg/l and 28 mg/l in LB culture, respectively. Taken together, our data suggest that pT7MT can be applied as a useful expression vector for stable and high-yield production of fusion proteins.     

Expression of a mutant regulatory subunit of cAMP-dependent protein kinase in the Caco-2 human colonic carcinoma cell line.

Caco-2 human colonic carcinoma cells were transfected with an expression vector encoding a mutant form of RI (regulatory subunit of the type 1 cAMP-dependent protein kinase), driven by the metallothionein 1 promoter. A stable transformant was isolated that expressed the mutant RI gene in a Zn(2+)-inducible manner. The consequences of the RI mutation on cAMP-dependent protein kinase activity, cell division, and regulation of chloride efflux were examined. When grown in the absence of ZnSO4, protein kinase activity in the transformant was stimulated 2.5-fold by cAMP and approached the levels of cAMP-dependent protein kinase activity seen in parental Caco-2 cells; when treated with ZnSO4, cAMP-dependent protein kinase activity in the transformant was inhibited by 60%. In the absence of ZnSO4 the transformant grew with the same doubling time and to the same saturation density as the untransformed parent. In the presence of ZnSO4 the transformant exhibited a cAMP-reversible inhibition of cell division, indicating that a functional cAMP-dependent protein kinase was required for the growth of these cells in culture. Induction of the mutant RI gene also abolished forskolin-stimulated chloride efflux from these cells, suggesting obligatory roles for cAMP and cAMP-dependent protein kinase in forskolin's actions on chloride channel activity. We anticipate that this transformant will be useful for further studies on the roles of cAMP and cAMP-dependent protein kinase in the regulation of intestinal epithelial cells, including regulation of cell proliferation and differentiation, and regulation of chloride channel activity by neurohormones and neurotransmitters.     

枯草芽孢杆菌α乙酰乳酸脱羧酶基因在啤酒酵母工业菌株中的表达

啤酒酿造中,双乙酰是影响啤酒生产熟化期长短及其风味的主要因素.存在于多种细菌中的a-乙酰乳酸脱羧酶(EC4.1.1.5,简称a-ALDC)[1]能将双乙酰的前体a-乙酰乳酸直接转化为对啤酒风味没有影响的乙偶姻,从而大大降低啤酒中双乙酰的含量,缩短啤酒熟化期.但所有的啤酒酵母菌不产生此酶.虽然在发酵过程中添加此酶是一个解决的途径,但解决问题的根本是将ALDC基因引入到啤酒酵母菌中.国外已开展这方面的研究[2,3],本研究组曾用随机克隆的方法获得了枯草芽孢杆菌a-ALDC基因[4],本文报道了枯草芽孢杆菌ALDC基因在工业用啤酒酵母中的表达研究结果.     

Characterization of hygromycin-resistant transformants of thermophilic fungus Thermomyces lanuginosus

Hygromycin-resistant stable transformants of the thermophilic fungus, Thermomyces lanuginosus, were obtained by electroporation of germinating aleurospores with a plasmid pMP6, coding for hygromycin resistance. Southern hybridization analysis revealed that the gene is integrated into the chromosome. The hygromycin-resistant transformants were characterized for morphological changes, growth response towards the presence of antagonistic metabolites (hygromycin, 2-deoxy-D-glucose, cylcoheximide, benlate and acriflavine) on plates and enzyme production (amylases, pectinases and xylanase) in shake flask cultures. A hygromycin-resistant transformant hyg 33 was characterized as non-sporulating, 2-deoxy-D-glucose-resistant, acriflavine-sensitive and xylanase hypo-producer and is being used as parental strain for breeding strains through protoplast fusion.