Molecular cloning of MER-2, a human chromosome-11-encoded red blood cell antigen,using linkage of cotransfected markers

We report the molecular cloning of a human gene MER-2located on chromosome 11 that encodes a cell surface antigen which is polymorphic on red blood cells. An essential element of the cloning strategy was cotransfection-induced linkage of pSV2-neo, which encodes resistance to the antibiotic G418, to the human MER-2gene. An important feature of the pSV2-neo construct is that the same gene (the transposon, Tn5) that encodes G418 resistance in eukaryotic cells confers neomycin resistance in bacteria. Chinese hamster ovary (CHO) cells were cotransfected with pSV2-neo and genomic DNA from a CHO ×human cell hybrid containing a single human chromosome (chromosome 11). Transfectants expressing both the human MER-2gene and G418 resistance were isolated by selection in the antibiotic G418, followed by indirect immunofluorescence using the monoclonal antibody 1D12, which recognizes the MER-2 antigen, manual enrichment, and single-cell cloning. Genomic DNA from a primary transfectant positive for MER-2expression and G418 resistance was used to construct a cosmid library and cosmid clones able to grow in neomycin were isolated. Of 150,000 cosmid clones screened, 90 were resistant to neomycin and of these, 11 contained human repetitive sequences. Five neomycin-resistant cosmid clones containing human repetitive DNA were able to transfect CHO cells for G418 resistance and MER-2expression.     

Characterization of the integration site of the CMV mtr in a tumor cell line

Previous studies have shown that a 558-bp fragment of human cytomegalovirus (CMV) DNA contained within pCM4127 and designated CMV mtr can morphologically transform rodents cells in vitro. By cotransformation with pCM4127 and a plasmid conferring G418 resistance, pSV2neo, morphologically transformed NIH3T3 cell lines were isolated. Dot blot hybridization indicated that approximately 30% of the transformants retained CMV sequences. Two cell lines which retained viral DNA were chosen for further study. They were capable of anchorage independent growth and formed tumors in nude mice. Integrated viral sequences in the transformants and tumor cell lines were analyzed by Southern blotting. A bacteriophage lambda library was constructed using a tumor cell line which retained a single copy of the viral sequences, and a phage was isolated which contained the integrated plasmid and the flanking cellular sequences. A complex rearrangement between pCM4127 and pSV2neo had occurred. DNA sequence analysis showed integration of the plasmid sequences into repetitive mouse DNA and identified an adjacent mouse sequence.     

Expression of the herpes simplex virus type-2 major DNA-binding protein (ICP8) gene in COS-1 cells

By the use of an SV40 origin of replication plasmid vector and the COS-1 cell system, expression of the gene encoding the herpes simplex virus type-2 (HSV-2) major DNA binding protein (ICP8) has been achieved. The HSV-2 4.5 kb BgIII 0 DNA fragment containing the ICP8 coding region was inserted into the plasmid vector pSVOd containing the SV40 origin of replication. Transfection of COS-1 cells by the resultant recombinant plasmid (pSVOd20), and subsequent multiplication of this plasmid, led to the production of the HSV-2 major DNA binding protein in sufficient quantities to allow its detection with either monoclonal or polyclonal antibodies as well as sera taken from HSV-2 patients.     

Identification of human gene complementing ts AlS9 mouse L-cell defect in DNA replication following DNA-mediated gene transfer

The temperature-sensitive (ts) mouse L-cell, ts AlS9, is defective in a gene required for nuclear DNA replication early in the S phase of the cell cycle. Human DNA sequences were introduced into ts AlS9 cells together with the plasmid pSV2neo, which can confer resistance to the drug geneticin. Cotransformants, expressing both the plasmid-derived neomycin gene and the transferred human AlS9 gene, were selected for growth in the presence of the drug at the nonpermissive temperature (npt). The resulting transformants retained a common set of human-specific Alu repetitive DNA sequences. These are likely to be accommodated within, or in proximity to, the transferred human AlS9 gene. The results obtained provide the basis for cloning human genes required for DNA replication.     

High efficiency transfection of monkey kidney COS-1 cells

Summary Methods are described for achieving high efficiency transient transfection of COS-1 cells. A transfection solution of vector DNA and DEAE-dextran in phosphate buffered saline is added to the cells, followed by treatment with culture medium containing chloroquine, resulting in a maximum efficiency of about 50%. The high efficiency obtained is primarily dependent on the use of Ca²⁺- or Mg²⁺-free buffer solutions for transfection, because the addition of these ions greatly reduces efficiency. Shocking the cells with dimethylsulfoxide does not increase transfection efficiency. COS-1 cells are monkey kidney cells that have a genomic insertion of the SV40 T antigen gene, allowing plasmid expression vectors bearing an SV40 replication origin to be amplified in these cells. Therefore this transfection method is useful for optimizing transient expression of genes in a mammalian cell system.     

High spontaneous mutation frequency of BPV shuttle vector

A recombinant shuttle vector containing the entire bovine papillomavirus (BPV) genome, sequences from pBR322, and the Escherichia coli gpt gene was used to transform mouse C127 cells. Plasmid extracted from the transformed mouse cells was used to transform a Gpt^– derivative of E. coli HB101, and the relative frequency of plasmids carrying a mutation in the gpt gene was determined. Approximate mutant frequencies of 3-16×10^–3 were observed for plasmid molecules which had been passaged through the mammalian cells. Restriction digest analysis indicated that most of the mutant plasmid molecules had gross rearrangements in their DNA structures.     

Somatic cell hybridization of Roberts syndrome and normal human fibroblasts transfected with plasmids carrying dominant selection markers

Roberts syndrome (RS) is a rare human recessive disorder involving, in the chromosomes of some patients, a characteristic puffing or splitting apart of the constitutive heterochromatin (the RS effect). We carried out somatic cell hybridizations between an RS cell strain (R22) with the heterochromatin abnormality and a hypoxanthine phosphoribosyltransferase-deficient cell strain (GM1662) with normal chromosome structure to determine if the presence of the normal genome would correct the RS effect in the hybrid cells. In order to provide the fibroblast strains with dominant selection markers for the hybridizations, GM1662 was transfected with the plasmid pSV3neo which conferred resistance to the antibiotic G418, and R22 was transfected with the plasmid pSV3gpt which provided resistance to mycophenolic acid. Two somatic cell hybridizations were carried out: (1) R22×GM1662 pSV3neo and (2) R22 pSVgpt×GM1662 pSV3neo. The RS effect was found to be absent in 95% and 92%, respectively, of the 200 hybrid cells examined in each experiment. This indicated that the GM1662 genome was able to correct the RS effect. The presence of the RS effect in a few of the hybrid cells was attributed to the unstable karyotype resulting from pSV3 transfection which presumably caused the loss of the normal allele(s) of the RS gene in these hybrid cells.     

Characterization of human folylpolyglutamate synthetase expressed in Chinese hamster ovary cells

Chinese hamster AUX B1 cells lack the enzyme folylpolyglutamate synthetase (FPGS) responsible for adding polyglutamates to folie acid. The human gene for FPGS was introduced into AUX B1 cells by cotransfection with human genomic DNA and either pSV2neo or pSV2gpt plasmid DNA. Cells were coselected for FPGS expression by growth in the absence of glycine, adenosine, and thymidine, and for drug resistance by growth in geneticin or mycophenolic acid. The presence of human enzyme in transfected cells was identified by relative enzyme activity determinations with ATP or dATP as substrates. The human HeLa cell enzyme displayed a 50% higher activity with dATP, and the hamster enzyme showed a 50% higher activity with ATP. Hamster and human enzymes also differed in the chain length of polyglutamates on cellular folate. Only monoglutamates were detected in AUX B1 cells, while wild-type hamster cells had predominantly hexa- and heptaglutamates, and human HeLa cells had predominantly hepta- and octaglutamates. Transformants with FPGS activity that showed a human enzyme preference for dATP also had folate polyglutamate chain lengths characteristic of the human enzyme.     

Differential Effects of Myeloperoxidase-Derived Oxidants on Escherichia coli DNA Replication

The microbicidal myeloperoxidase (MPO)-H₂O₂-chloride system strongly inhibits Escherichia coli DNA synthesis. Also, cell envelopes from MPO-treated E. coli cells lose their ability to interact with hemimethylated DNA sequences of oriC, the chromosomal origin of replication, raising the prospect that suppression of DNA synthesis involves impairment of oriC-related functions (H. Rosen, et al. Proc. Natl. Acad. Sci. USA, 87:10048–10052, 1990). To evaluate whether origin-specific DNA sequences play a role in the MPO effect on E. coli DNA synthesis, plasmid DNA replication was compared to total (chromosomal) DNA replication for six plasmids with three distinct origins of replication. Plasmid pCM700 replication, replicating from oriC, was as sensitive to MPO-mediated inhibition as was total (chromosomal) DNA replication. A regression line describing this relationship had a slope of 0.90, and the r² was 0.89. In contrast, the replication activities of three of four non-oriC plasmids, pUC19, pACYC184, and pSC101, demonstrated significant early resistance to inhibition by MPO-derived oxidants. The exception to this resistance pattern was plasmid pSP102, which has an origin derived from P1 phage. pSP102 replication declined similarly to that of total DNA synthesis. The regression line for pSP102 replication versus total DNA synthesis had a slope of 0.95, and the r² was 0.92. The biochemical requirements for P1-mediated replication are strikingly similar to those for oriC-mediated replication. It is proposed that one of these requirements, common to oriC and the P1 origin but not critical to the replication of the other non-oriC plasmids, is an important target for MPO-mediated oxidations that mediate the initial decline in E. coli chromosomal DNA synthesis.     

Rescue of aDrosophila temperature-sensitive mutant cell line by DNA transfection

At the nonpermissive temperature, the shibire^ts gene of Drosophilacauses adult paralysis and cell death during development. We have used DNA-mediated gene transfer with wildtype genomic DNA to rescue the lethal phenotype of shi^ts cultured cells and have isolated cell lines which grow at the nonpermissive temperature of 29° C. By using restriction endonucleases, we confirm that this rescue is DNA sequence dependent. Further, cells cotransfected with wildtype genomic DNA and plasmid DNA contain plasmid sequences when selected for survival at 29° C. This confirms the uptake of DNA by the transformed cells and provides a simple temperature selection technique allowing coselection of genes which cannot themselves be selected for.     

The effect of canavanine on the maintenance in yeast of chimeric plasmids containing portions of the 2-µm DNA plasmid

Summary We have found that the application of the amino acid analog canavanine to a culture of yeast cells transformed with chimeric plasmids based on the yeast 2-µm DNA plasmid increases the percentage of cells which have lost the transforming plasmid. This effect is found whether the plasmid carries the CAN1 sensitive allele and the yeast strain carries a can1 mutation confering resistance, or the plasmid contains no CAN1 allele and the yeast strain carries the wild-type CAN1 sensitive allele. Canavanine exerts this effect on yeast strains transformed with chimeric plasmids containing either a portion or the entire 2-µm DNA plasmid, yet canavanine does not appear to effect the maintenance of the native 2-µm DNA plasmid complement within the cell. The effect of canavanine on strains transformed with chimeric plasmids is the same whether or not the yeast strain also contains native 2-µm plasmid DNA. Neither the amino acid analog ethionine, the protein synthesis inhibitor cycloheximide, nor the DNA replication inhibitor hydroxyurea exhibit this effect. Some of the experimental results suggest that canavanine may be a curing agent rather than an agent which selects for spontaneous plasmid loss.     

Titration of replication activity by increasing ARS dosage in yeast plasmids

The rep1 region of the yeast mitochondrial genome, a putative replication origin, contains a weak autonomously replicating sequence (ARS). Nucleotidesequence and deletion analyses have identified two 11-base pair ARS consensus sequences, numerous near matches to the ARS core, and a region of curvature that may contribute to ARS function. Based on the amplified nature of petite-derivative mitochondrial DNA encompassing this locus, we have constructed plasmids containing an increasing dosage of ARS elements. The rep1 ARS element can have an additive effect on plasmid stability when present either as a tandem dimer or as an unlinked pair. However, the presence of a third ARS copy does not further enhance plasmid stability. These results indicate that measurable dosage effects can be defined only in circumstances where weak ARS elements are employed, and that plasmid maintenance within yeast cells is saturable and varies among the different sequences promoting replication.     

Establishment of transformed swine fibroblast cell lines using SV40 large T antigen

Summary Swine testicle cell lines were established by transformation of primary swine testicle (PST) cells with an SV40 plasmid (pSV3-neo), which contains genes conferring resistance to neomycin and expressing SV40 large T antigen. Plasmid DNA was transfected into PST cells using a lipofection system. Two related plasmids, pSV2-neo and pSV5-neo, failed to induce transformed cells. Cells transformed with pSV3-neo formed single colonies that were resistant to the antibiotic, G418, and expressed large T antigen. Upon two cycles of cloning by endpoint dilution method, three transformed clones, designated transformed swine testicle (tST)-3, tST-14 and tST-18, were selected and characterized in regards to cell replication and susceptibility to swine viruses. The resultant clones were compared with a counterpart non-transformed ST cell line (ATCC-ST). The three tST cell lines showed longer or the same doubling times and higher saturation densities compared to ATCC-ST cells. These cells were free from a range of adventitious agents and supported the replication of porcine parvovirus (PPV), pseudorabies virus (PRV) and transmissible gastroenteritis virus (TGEV), comparable to ATCC-ST cells. All three cell lines have been maintained in continuous cultures for over 60 passages with no changes in growth characteristics. These findings indicate that lipofection with pSV3-neo is an efficient means for the introduction of exogenous DNA into porcine cells and for establishment of transformed immortalized cell lines.     

A genetically engineered V79 cell line SD1 expressing rat CYP2B1 exhibits chromosomal instability at the integration site of the transfected DNA

The genetically engineered cell line SD1 was constructed byco-transfection of V79 Chinese hamster cells with two plasmids:one containing a full-length cDNA encoding rat CYP2B1 and thesecond incorporating a selective marker gene. This cell linehas been used in gene mutation tests and in cytokinesis-blockmicronucleus assays to identify procarcinogens which are metabolizedby CYP2B1 to reactive metabolites. An elevated frequency ofspontaneous micronuclei was recorded in SD1 cells compared toparental V79 cultures. Karyotypic analysis revealed a chromosomalinstability which was manifested by amplification of the p-armsof a chromosome designated ‘n’ (derived from chromosome8). This chromosome was variable in length and sometimes exhibiteda telomeric fusion which led to the formation of a dicentricchromosome. Fluorescence in situ hybridization with digoxigenin-labelledplasmid DNA showed the presence of pSV450 plasmid DNA co-amplifiedwith genomic DNA sequences located in the terminal region ofchromosome ‘n’. ¹Present address: Molecular Genetics Laboratory, Departmentof Pathology, Royal Devon and Exeter Healthcare NHS Trust, BarrackRoad, Exeter EX2 5DW, UK. ²To whom correspondence should be addressed      

Evidence for autonomous replication and stabilization of recombinant plasmids in the transformants of yeast Hansenula polymorpha

Summary For the transformation of the yeast Hansenula polymorpha we have constructed a set of hybrid plasmids carrying the LEU2 gene of Saccharomyces cerevisiae as a selective marker and fragments of mitochondrial DNA of Candida utilis and H. polymorpha or chromosomal DNA fragments of H. polymorpha as replicator sequences. The replication properties of chimeric plasmids in the yeast H. polymorpha were investigated. We showed that for plasmids propagated autonomously in this yeast the plasmid monomers could be detected in the transformants only during the immediate time after the transformation event. Further growth under selective conditions led to the selection of polymeric forms of plasmid DNA as it was clearly shown for transformants carrying cosmid pL2 with mtDNA fragment of C. utilis. Such transformants carrying polymerized plasmids showed a remarkably increased stability of the transformed phenotype. Cosmid pL2 was able to shuttle between Escherichia coli, S. cerevisiae and H. polymorpha, whereas plasmids with DNA fragments from H. polymorpha did not transform S. cerevisiae effectively.     

Transfection of lymphoblastoid cells using DNA-loaded reconstituted Sendai virus envelopes: Expression of transfected DNA and selection of transfected cells

The American Burkitt's lymphoma cell line Loukes was cotransfected with cloned BamHI K fragment of EBV DNA and a vector pSV2neo. Reconstituted Sendai virus envelopes (RSVE) loaded with DNA were used for efficient gene transfer. Two cell lines have been obtained following culture in the presence of geneticin sulfate (G-418). Messenger RNA from both transfected DNAs was expressed during the whole period of observation, 42 days after transfection. This method provides a relatively simple and efficient means for selection of lymphoblastoid cells expressing a transfected gene.     

Enhanced transfection of a bacterial plasmid into hybridoma cells by electroporation: application for the selection of hybrid hybridoma (quadroma) cell lines.

A procedure was investigated to transduce a bacterial plasmid containing a specific drug resistance marker (pSV2-neo), into a hybridoma cell line using electroporation. The effect of several buffers and the form of plasmid DNA (circular or linearized) on the stable transfection frequency were examined. When complete cell culture medium (DMEM) was used as electroporation buffer, we observed a two-fold increase in post-pulse viability and a ten- to thirty-fold increase in the transfection frequency of pSV2-neo, as compared with HEPES buffered 0.15 M sodium chloride. Supplementing DMEM with fetal bovine serum (DMEM + FBS) had some beneficial effect on post-pulse viability of the cells after electroporation, but did not markedly increase stable transfection frequency as compared with DMEM alone. Furthermore, with DMEM + FBS, the intact plasmid was transfected as effectively as linearized PSV2-neo. However, when using HEPES buffered saline, the transfection frequency of pSV2-neo increased two-fold after linearization as compared with intact plasmid. The drug resistance was used successfully as a marker for the selection of hybrid hybridoma (quadroma) cell lines after fusing two different hybridoma cell lines, producing anti-fibrin and anti-plasminogen activator antibodies respectively. The quadroma cells produced bispecific antibodies that are capable of accumulating plasminogen activator on a fibrin surface.     

The in vivo clearance of Ha-ras transformants by natural killer cells

The experiments in this study were designed to test the hypothesis that natural killer (NK) cells play a role in host surveillance against early neoplastic changes in the malignant process. C3H 10T1/2 mouse fibroblasts were transfected with a pSV2-neo plasmid vector which contains EJ, the mutated c-Ha-ras, regulated by its own promoter. Control cells were transfected with pSV2-neo alone and did not contain the ras gene. Oncogene-transfected cells were compared with control cells for lung colony formation following tail vein injection into C3H mice. Intravenous injection of ras-transfected 10T1/2 cells induced marked lung colony formation in vivo, whereas C3H 10T1/2 parental lines or 10T1/2 cells transfected with pSV2-neo alone induced no lung colonies in C3H mice. The colonising potential of ras transfectants could be decreased by augmentation of NK activity by injection of polyinosinic cytidylic acid and increased by depletion of NK effectors with anti-asialo GM₁. Experiments with beige mice demonstrated that the mortality of syngeneic, NK-deficient C3H-bg/bg mice injected with ras tranfectants was significantly greater than similarly treated NK-normal C3H-+/bg littermate controls. The results support the view that NK cells are capable in vivo of recognizing early defined stages in the neoplastic process initiated by oncogenes.     

Transformations of Penicillium islandicum and Penicillium frequentans that produce anthraquinone-related compounds

Wild-type strains of Penicillium islandicum and Penicillium frequentans, which produce anthraquinone and related compounds, were transformed to benomyl and hygromycin B resistance. Plasmids pSV50 and pBT6, with benomyl-resistant -tublin genes, and plasmids pAN7-1 and pDH25, with a bacterial hygromycin phosphotransferase gene under the control of Aspergillus nidulans sequences, were used respectively. Transformation frequencies with these plasmids were 10–20 transformants per g of DNA per 4-8×10⁷ viable protoplasts. Intergration of plasmid DNAs into chromosomal DNAs was confirmed by Southern-blot analysis. Copy numbers and sites of integration varied among transformants. The integrated plasmid DNAs conferring a drug-resistant phenotype were mitotically stable with or without selection. The demonstration of such transformation systems is the essential first step in the application of recombinant DNA technology to study the biosynthetic genes of anthraquinone and related compounds in P. islandicum and P. frequentans.