Metabolic burden in recombinant CHO cells: effect ofdhfr gene amplification andlacZ expression

Foreign protein production levels in two recombinant Chinese hamster ovary (CHO) cell lines were compared in cells transfected with different expression vectors. One vector pNL1 contained the gene for neomycin resistance (neo ^r ) and thelacZ gene which codes for intracellular -galactosidase, with both genes controlled by the constitutive simian virus (SV40) promoter. The other vector CDG contained the amplifiabledhfr gene andlacZ gene, controlled by the constitutive SV40 and cytomegalovirus (CMV) promoters, respectively. Cell growth and -galactosidase expression were compared quantitatively after cells were selected in different concentrations of the neomycin analog G418 and methotrexate, respectively. A 62% reduction in growth rate occurred in recombinant CHO cells in which thelacZ anddhfr genes were highly amplified and expressed. In contrast, the combined effects of the unamplifiedneo ^r gene andlacZ gene expression on the growth kinetics were small. Any metabolic burden caused bylacZ gene expression, which was evaluated separately from the effect ofneo ^r gene expression, must be negligible, as higher expression of -galactosidase (1.5×10^–6 units/cell) occurred in unamplified cells compared to the cells in whichlacZ was amplified by thedhfr-containing vector (3×10^–7 units/cell). Thus, the main factor causing severe growth reduction (metabolic burden) in cells containing the amplifieddhfr gene system was not overexpression of -galactosidase butdhfr andlacZ gene co-amplification anddhfr gene expression.     

Induction of Antibiotic Resistance in Paramecium tetraurelia by the Bacterial Gene APH-3'-II

We have generated a transformation marker for Paramecium using a Paramecium expression vector (pPXV) and the open reading frame (ORF) of the bacterial antibiotic resistance gene aminoglycoside 3'-phosphotransferase-II (APH-3'-II or neo^r) from the transposon Tn5. The expression vector contained a small multiple cloning site between the 5' and 3' non-coding regions of the calmodulin gene, and Tetrahymena telomere sequences for the stability of the plasmid in Paramecium. After the neo^r ORF was inserted, the plasmid was referred to as pPXV-NEO. Delivery of approximately 10–20 picoliters of linearized PXV-NEO at > 2000 copies/pl into the macronucleus effected 100% transformation. Southern and Northern blot hybridization showed the presence of neo^r-specific DNA and RNA, respectively, in all of the transformed clones but not in the untransformed clones. The degree of resistance to G-418, and the concentrations of neo^r-specific DNA and neo^r-specific RNA in the clones were proportional to the concentration of the vector injected. We have demonstrated that when the linearized plasmid was injected into the macronucleus, the prokaryotic sequence conferred an antibiotic resistance to Paramecium despite codon-usage differences.     

Apparent gene conversion in an Escherichia coli rec+ strain is explained by multiple rounds of reciprocal crossing-over

Summary Gene conversion, the non-reciprocal transfer of sequence information between homologous DNA sequences, has been reported in lower eukaryotes, mammals and in Escherichia coli. In an E. coli rec ⁺ strain, we established a plasmid carrying two different deleted neo genes (neoDL and neoDR) in an inverted orientation and then selected for homologous recombination events that had reconstructed an intact neo ⁺ gene. We found some plasmids that had apparently experienced intramolecular gene conversion. Further evidence, however, suggests that they are products of multiple rounds of reciprocal crossing-over,apparently involving two plasmid molecules. First, most of the Neo⁺ clones contained multiple types of Neo⁺ plasmids, although the frequency of producing the neo ⁺ clones was low. Second, all the neo ⁺ clones also contained, as a minority, one particular form of dimer, which can be formed by reciprocal crossing-over between neoDL of one plasmid molecule and neoDR of another plasmid molecule. Third, in reconstruction experiments, we cloned and purified this dimer and transferred it back into the rec ⁺ cells. The dimer gave rise to clones containing multiple types of neo ⁺ recombinant monomers, including those apparent gene conversion types, and containing only few molecules of this dimer plasmid.     

Efficient expression of small RNA polymerase III genes from a novel simian virus 40 vector and their effect on viral gene expression.

In the past, simian virus 40 (SV40) has been used as a cloning vehicle to clone foreign genes by substituting portions of the viral genome vital for viral replication. Propagation of these defective viruses required a helper virus and the recombinant viruses obtained could be grown only as a mixture. In this study, we describe a novel nondefective SV40 vector to clone small RNA polymerase III genes. Two small RNA polymerase III genes, an amber suppressor human serine tRNA gene and the adenovirus (Ad) VAI RNA gene, were cloned in the intron region of the large-T antigen gene of SV40 after deleting DNA sequences coding for the small-t polypeptide. The recombinant viruses grew to wild type levels and showed no growth defects. When CV-1p cells were infected with these viruses, the cloned RNA polymerase III genes were expressed at high levels at late times. Interestingly, large amounts VAI RNA in CV-1p cells infected with SV40-VA recombinant virus, did not enhance translation of viral mRNAs significantly but did lead to a 3 to 4 fold increase in the steady state levels of large-T mRNA suggesting a novel function for VAI RNA in SV40 infected monkey cells. Furthermore, VAI mutants which fail to function in Ad infected human cells also failed to enhance the levels of large-T mRNAs in monkey cells infected with SV40. The simple SV40 vector described here may be useful to study the structure and function of small RNA polymerase III genes in the context of a eucaryotic chromosome. In addition, the nondefective recombinant SV40 which expresses the suppressor tRNA gene at high levels may provide a useful helper system to propagate animal viruses with amber mutations in essential genes.     

Active human erythropoietin expressed in insect cells using a baculovirus vector: A role for N-linked oligosaccharide

Biologically active recombinant human erythropoietin has been expressed at high levels in an insect cell background. Expression involved the preparation of a human erythropoietin cDNA, the transfer of this cDNA to the Autographa californica nuclear polyhedrosis virus (AcNPV) genome under the polyhedrin gene promoter, and the subsequent infection of Spodoptera frugiperda cells with recombinant AcNPV. Erythropoietin cDNA was prepared through the expression of the human erythropoietin gene in COS cells using pSV2 and the construction of a COS cell cDNA library in bacteriophage Lambda GT10. Prior to transfer to the AcNPV genome, erythropoietin cDNA isolated from this library was modified at the 3′-terminus in order to replace genomic erythropoietin for SV40 cDNA derived from pSV2. Transfer of this cDNA to AcNPV and the infection of S. frugiperda cells with cloned recombinant virus led to the secretion of erythropoietin: based on bioassay, rates of hormone secretion (over 40 U/ml per h) were 50-fold greater than observed for COS cells. The purified recombinant product possessed full biological activity (at least 200000 U/mg), but was of lower M_r (23000) than human erythropoietin produced in COS cells (30000) or purified from urine (30000 to 38000). This difference was attributed to the glycosylation of erythropoietin in S. frugiperda cells with oligosaccharides of only limited size. Further removal of N-linked oligosac-charides from this M_r 23000 hormone using N-Glycanase yielded an apo-erythropoietin (M_r 18000) which possessed substantially reduced biological activity. These results indicate that glycosylation, but not the normal processing of oligosaccharides to complex types, is required for the full hormonal activity of human erythropoietin during red cell development.     

Transformation of Mucor circinelloides with Autoreplicative Vectors Containing Homologous and Heterologous ARS Elements and the Dominant Cbx r Carboxine-Resistance Gene

Mucor circinelloides transformants prototrophic to leucine and resistant to carboxine (Leu⁺ Cbx^r) have been obtained by treatment of protoplasts with plasmid constructs containing homologous leuA gene and adjacent autonomously replicating sequences (ARS) element combined with the Cbx^r(carboxine-resistance) gene of Ustilago maydis and ARS sequences from this basidiomycete (plasmid pGG37) or from the 2 μ plasmid of Saccharomyces cerevisiae (plasmid pGG43). The presence in the same plasmid molecule of the M. circinelloides leuA gene and adjacent ARS element together with heterologous ARS elements produced an increase in the transformation frequency of about 65–120%. The presence of autoreplicating plasmid molecules in the transformants was demonstrated by mitotic stability experiments, by Southern analysis, and by the rescue of plasmids from transformed bacterial cells.     

Cloning and expression of a xylanase gene from Clostridium acetobutylicum P262 in Escherichia coli

Summary A xylanase gene from Clostridium acetobutylicum P262 was cloned on a recombinant plasmid pHZ300 which enabled Escherichia coli HB101 cells to produce intracellular xylanase activity. The xylanase gene was located on a 2 kb DNA fragment. The cloned xylanase had an apparent M _r of approximately 28 000 and an isoelectric point of approximately 10. Optimum xylanase activity was obtained at pH 6.0 at 37–43° C. Comparison with a xylanase partially purified from the culture medium of C. acetobutylicum P262 showed that the enzymes had similar characteristics and western blot analysis showed cross-reactivity between antibodies raised against the purified cloned enzyme and a polypeptide of the same M _r from C. acetobutylicum P262.     

Identification of new peptides synthesized under the hydrogenase control system of Alcaligenes eutrophus

Total protein of Alcaligenes eutrophus was analyzed by two-dimensional protein map. Cells grown at 30° C expressed hydrogen-oxidizing (Hox) ability mediated by a soluble (Hos) and a particulate hydrogenase (Hop). Hox ability was not expressed at 37° C (HoxTs). The six subunits of the two hydrogenases were identified. Besides these six subunits eight peptides were not or hardly detected at 37° C. The mutant HF117 which expressed Hox ability at 37° C (HoxTr), formed the hydrogenase peptides and five of the eight peptides. These peptides designated B, C, E, F, and H were characterized by their isoelectric point and molecular mass (M _r); their M _r were 18 800, 45 400, 41 900, 39 400, and 40 600, respectively. The five peptides were not formed in regulatory Hox^– mutants, and not formed in mutants cured of plasmid pHG1, carrying the genetic information for hydrogenase formation. Strain HF160, carrying a Tn5 insertion in a gene essential for Hos expression specifically did not form the B-peptide. All peptides were found in the soluble fraction of cell extracts, the F-peptide was also detected in the particulate fraction. The function of the new Hox-peptides is presently unknown.Abbreviations PAGE polyacrylamide gelelectrophoresis - SDS sodium dodecylsulfate - Hox hydrogen oxidizing ability     

A genetic system to detect mitotic recombination between repeated chromosomal sequences in Drosophila Schneider line 2 cells

In order to study mitotic homologous recombination in somatic Drosophila melanogaster cells in vitro and to learn more on the question how recombination is influenced by mutagens, a genetic system was developed where spontaneous and drug-induced recombination could be monitored. Two recombination reporter substrates were stably introduced in multiple copies into the genome of established D. melanogaster Schneider line 2 cells: one plasmid (pSB310) contained the 5′ and 3′ deleted neomycin phosphoribosyltransferase alleles neoL and neoR as direct repeats; the other (pSB485) contained similar deletions (lacZL and lacZR) of the β-galactosidase gene (lacZ). Restoration of a functional neo gene upon mitotic recombination between homologous sequences allowed direct selection for the event, whereas recombination in single cells harbouring the integrated lacZ-based reporter plasmid was detected by histochemical staining or flow cytometric analysis (FACS). The neo-based construct in the clonal transgenic cell line 44CD4 showed a spontaneous recombination frequency of 2.9×10⁻⁴, whereas the 485AD1 cell line harbouring the lacZ-based construct exhibited a frequency of 2.8×10⁻⁴. The alkylating agents EMS and MMS and the clastogen mitomycin C were able to induce recombination in the 485AD1 cell line in a dose-dependent manner. The results obtained from these studies suggest that the transgenic cell lines are potentially useful tools for identifying agents which stimulate direct repeat recombination in somatic Drosophila cells.     

Gene conversion in the Escherichia coli RecF pathway: a successive half crossing-over model.

Summary Gene conversion - apparently non-reciprocal transfer of sequence information between homologous DNA sequences - has been reported in various organisms. Frequent association of gene conversion with reciprocal exchange (crossing-over) of the flanking sequences in meiosis has formed the basis of the current view that gene conversion reflects events at the site of interaction during homologous recombination. In order to analyze mechanisms of gene conversion and homologous recombination in an Escherichia coli strain with an active RecF pathway (recBC sbcBC), we first established in cells of this strain a plasmid carrying two mutant neo genes, each deleted for a different gene segment, in inverted orientation. We then selected kanamycin-resistant plasmids that had reconstituted an intact neo ⁺ gene by homologous recombination. We found that all the neo ⁺ plasmids from these clones belonged to the gene-conversion type in the sense that they carried one neo ⁺ gene and retained one of the mutant neo genes. This apparent gene conversion was, however, only very rarely accompanied by apparent crossing-over of the flanking sequences. This is in contrast to the case in a rec ⁺ strain. or in a strain with an active RecE pathway (recBC sbcA). Our further analyses, especially comparisons with apparent gene conversion in the rec ⁺ strain, led us to propose a mechanism for this biased gene conversion. This successive half crossing-over model proposes that the elementary recombinational process is half crossing;-over in the sense that it generates only one recombinant DNA duplex molecule, and leaves one or two free end(s), out of two parental DNA duplexes. The resulting free end is, the model assumes, recombinogenic and frequently engages in a second round of half crossing-over with the recombinant duplex. The products resulting from such interaction involving two molecules of the plasmid would be classified as belonging to the gene-conversion type without crossing-over. We constructed a dimeric molecule that mimics the intermediate form hypothesized in this model and introduced it into cells. Biased gene conversion products were obtained in this reconstruction experiment. The half crossing-over mechanism can also explain formation of huge linear multimers of bacterial plasmids, the nature of transcribable recombination products in bacterial conjugation, chromosomal gene conversion not accompanied by flanking exchange (like that in yeast mating-type switching), and antigenic variation in microorganisms.     

小鼠白细胞介素33真核表达质粒的构建及表达

克隆小鼠IL-33基因构建其真核表达质粒,并转染COS-7细胞检测其表达。提取C57BL/6小鼠肺组织总RNA,经反转录聚合酶链式反应（RT-PCR）扩增小鼠IL-33基因,酶切后插入pcDNA^TM3.1/myc HisA构建其真核表达质粒pcDNA-3.1-IL-33,重组质粒转染COS-7细胞,RT-PCR和免疫印迹法（western blotting）检测目的基因表达。结果显示,pcDNA3.1-IL-33中插入的片段序列测定结果与小鼠IL-33cDNA序列一致,重组质粒转染COS-7细胞后检测到相应mRNA及蛋白表达。成功克隆了小鼠IL-33基因cDNA,并构建其真核表达质粒。     

Biological activity of paramyxovirus fusion proteins: factors influencing formation of syncytia.

The fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins of the paramyxovirus simian virus 5 (SV5) were expressed individually or coexpressed in CV-1 cells by using SV40-based vectors and recombinant vaccinia viruses. The extent of detectable fusion in a syncytium formation assay was found to be affected by the expression system used. In addition, when HN was coexpressed with F, it was found that the expression vector system influenced the contribution of HN in forming syncytia. The abilities of the SV5, human parainfluenza virus type 3, and Newcastle disease virus F glycoproteins to cause fusion, when expressed alone or coexpressed with HN, were directly compared by using the SV40-based vector system in CV-1 cells. The F proteins exhibited various degrees of fusion activity independent of HN expression, but the formation of syncytia could be enhanced to different extents by the coexpression of the homotypic HN protein.     

Human HOXA5 homeodomain enhances protein transduction and its application to vascular inflammation

Cellular protein delivery is an emerging technique by which exogenous recombinant proteins are delivered into mammalian cells across the membrane. We have developed an Escherichia coli expression vector including human specific gene sequences for protein cellular delivery. The plasmid was generated by ligation the nucleotides 770–817 of the homeobox A5 mRNA sequence which was matched with protein transduction domain (PTD) of homeodomain protein A5 (HOXA5) into pET expression vector. The cellular uptake of HOXA5-PTD-EGFP was detected in 1 min and its transduction reached a maximum at 1 h within cell lysates. The cellular uptake of HOXA5-EGFP at 37 °C was greater than in 4 °C. For study for the functional role of human HOXA5-PTD, we purified HOXA5-APE1/Ref-1 and applied it on monocyte adhesion. Pretreatment with HOXA5-APE1/Ref-1 (100 nM) inhibited TNF-α-induced monocyte adhesion to endothelial cells, compared with HOXA5-EGFP. Taken together, our data suggested that human HOXA5-PTD vector provides a powerful research tools for uncovering cellular functions of proteins or for the generation of human PTD-containing proteins.     

Introduction and recovery of a selectable bacterial gene from the genome of mammalian cells.

The simian virus 40 (SV40)-pBR322 recombinant, pSV2, carrying the origin of SV40 replication and the gpt gene of Escherichia coli, has been stably introduced into Chinese hamster ovary hprt- cells. All gpt-transformed cell lines were found to contain one or more insertions of pSV2 sequences exclusively associated with high-molecular-weight DNA. Additional analyses showed that at least one integrated copy in each cell line retained an intact gpt gene and flanking SV40 sequences required for expression of xanthine-guanine phosphoribosyltransferase. Most cell lines contained pSV2 sequences which had integrated with partial sequence duplication. Upon fusion with COS-1 cells, a simian cell line permissive for autonomous pSV2 replication, most gpt-transformed cell lines produced low-molecular-weight DNA molecules related to pSV2. The majority of these replicating DNAs were indistinguishable from the original transfecting plasmid in both size and restriction enzyme cleavage pattern. In addition, the recovered DNA molecules were able to confer ampicillin resistance to E. coli and to transform mouse L cells and Gpt- E. coli to a Gpt+ phenotype. These studies indicate that all of the genetic information carried by this SV40-plasmid recombinant can be introduced into and retrieved from the genome of mammalian cells.     

SV40-transformed simian cells support the replication of early SV40 mutants

CV-1, an established line of simian cells permissive for lytic growth of SV40, were transformed by an origin-defective mutant of SV40 which codes for wild-type T antigen. Three transformed lines (COS-1, -3, -7) were established and found to contain T antigen; retain complete permissiveness for lytic growth of SV40; support the replication of tsA209 virus at 40 degrees C; and support the replication of pure populations of SV40 mutants with deletions in the early region. One of the lines (COS-1) contains a single integrated copy of the complete early region of SV40 DNA. These cells are possible hosts for the propagation of pure populations of recombinant SV40 viruses.     

Stable co-transformation of maize protoplasts with gusA and neo genes

An efficient co-transformation protocol using polyethylene glycol was developed for Zea mays L. (cv. A188 × BMS) protoplasts isolated from suspension culture cells. Co-transformation was accomplished by using plasmid constructions containing -glucuronidase (gusA) or neomycin phosphotransferase (neo) gene coding sequences; both were under control of the CaMV 35S promoter. Protoplast culture and transformation conditions were optimized to assure efficient recovery of transformed cells. The overall efficiency of transformation was 1 × 10^–4 (calculated per viable protoplast plated). Among kanamycin-resistant lines, 50% showed a high level of GUS activity (above one unit). Southern blot hybridization confirmed the presence of numerous gusA and neo coding sequences in the maize genome. In two analyzed lines, integrated sequences appeared to be organized in tandem head-to-tail repeats. Results also indicated that the integrated sequences were partially methylated.