Bifunctional reporter genes: construction and expression in prokaryotic and eukaryotic cells

Bifunctional reporter proteins were constructed to combine Clostridium thermocellum lichenase (LicBM2) with Aequorea victoria green fluorescent protein (GFP) or with Escherichia coli beta-glucuronidase (GUS). The major properties of the initial proteins were preserved in the hybrid ones: LicBM2 was active at 65 degrees C, GFP fluoresced, and GUS hydrolyzed its substrates. LicBM2 remained active after extension of its C of N end. Bifunctional reporter systems were shown to provide a convenient tool for studying the gene expression regulation in prokaryotic (E. coli) and eukaryotic (Saccharomyces cerevisiae, mammalian) cells, advantages of one reporter compensating for drawbacks of the other.     

DNA-mediated transfer of actinomycin D resistance into cultured mouse cells

The transfer of high molecular weight (HMW) DNA isolated from Syrian hamster actinomycin D-resistant cells (C12TSV5/R) to sensitive A9 mouse cells, resulted in the growth in selective medium of transferent clones A9 ActR C11, C12 and C13). The resistant mouse cells have presumably acquired a gene(s) conferring actinomycin D resistance and express a 140-150 K surface glycoprotein, apparently involved in the drug resistance.     

Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells

Multiprotein complexes catalyze vital biological functions in the cell. A paramount objective of the SPINE2 project was to address the structural molecular biology of these multiprotein complexes, by enlisting and developing enabling technologies for their study. An emerging key prerequisite for studying complex biological specimens is their recombinant overproduction. Novel reagents and streamlined protocols for rapidly assembling co-expression constructs for this purpose have been designed and validated. The high-throughput pipeline implemented at IGBMC Strasbourg and the ACEMBL platform at the EMBL Grenoble utilize recombinant overexpression systems for heterologous expression of proteins and their complexes. Extension of the ACEMBL platform technology to include eukaryotic hosts such as insect and mammalian cells has been achieved. Efficient production of large multicomponent protein complexes for structural studies using the baculovirus/insect cell system can be hampered by a stoichiometric imbalance of the subunits produced. A polyprotein strategy has been developed to overcome this bottleneck and has been successfully implemented in our MultiBac baculovirus expression system for producing multiprotein complexes.     

Cloning of eukaryotic genes in single-strand phage vectors: the human interferon genes

Using oligonucleotide probes with defined sequences, we have selected clones from a human lymphocyte cDNA library which represent human leukocyte (HuIFN-α) and fibroblast (HuIFN-β) interferon gene sequences. Double-stranded f1 phage DNA was used as the vector for initial cloning of cDNA. Clones carrying interferon gene sequences were identified by hybridization with the oligonucleotide probes. The same oligonucleotide probes were used as primers for dideoxy chain termination sequencing of the clones. One HuIFN-α clone, 201, has a nucleotide sequence different from published HuIFN-α sequences. Under control of the lacUV5 promoter, the 201 gene has been used to express biologically active HuIFN-α in Escherichia coli.     

扎伊尔埃博拉病毒糖蛋白基因的克隆和表达

埃博拉病毒属丝状病毒科,能引发动物和人出血热症状,人感染后病死率高达90%以上,目前还没有有效预防和治疗的药物和疫苗。近年来,这种烈性传染病病毒传入我国的可能性不断加大,给我国公共卫生应急体系带来新的挑战。本研究针对埃博拉病毒的最主要结构蛋白——糖蛋白(GP),构建了重组原核表达载体pET28a(+)-GP1(33~313aa)、pET28a(+)-GP1(190~313aa)、pET28a(+)-GP2(502~632aa)、pET28a(+)-sGP,以及重组真核表达载体pcDNA3.1(+)-edited GP、pcDNA3.1(+)-GP1、pcDNA3.1(+)-GP。结果表明,GP1(33~313aa)、GP1(190~313aa)和sGP能在大肠埃希菌BL21(DE3)中以包涵体的形式表达,GP、GP1和GP2能在HEK293T细胞中表达,但均不能在BHK21细胞中表达。本研究为进一步探索埃博拉病毒GP的结构和功能及GP抗体制备奠定了基础。     

Transfer and expression of plasmids containing human cytomegalovirus immediate-early gene 1 promoter-enhancer sequences in eukaryotic and prokaryotic cells

The human cytomegalovirus immediate-early gene region 1 promoter-enhancer is active in bacteria and in many mammalian cells. Recombinant plasmids containing portions of this DNA can be used to promote the expression of foreign proteins in many cells. In this communication, we report the optimal conditions for transfer of plasmid DNA to cells by electroporation and the transient expression assays which document the activity of different promoter constructions. The observed activity of the human cytomegalovirus promoter is more than 100-fold higher than the activity of the early promoter of SV40.     

Functional expression of prokaryotic and eukaryotic genes in Escherichia coli for conversion of glucose to p-hydroxystyrene

The chemical monomer p-hydroxystyrene (pHS) is used for producing a number of important industrial polymers from petroleum-based feedstocks. In an alternative approach, the microbial production of pHS can be envisioned by linking together a number of different metabolic pathways, of which those based on using glucose for carbon and energy are currently the most economical. The biological process conserves petroleum when glucose is converted to the aromatic amino acid L-tyrosine, which is deaminated by a tyrosine/phenylalanine ammonia-lyase (PAL/TAL) enzyme to yield p-hydroxycinnamic acid (pHCA). Subsequent decarboxylation of pHCA gives rise to pHS. Bacteria able to efficiently decarboxylate pHCA to pHS using a pHCA decarboxylase (PDC) include Bacillus subtilis, Pseudomonas fluorescens and Lactobacillus plantarum. Both B. subtilis and L. plantarum possess high levels of pHCA-inducible decarboxylase activity and were chosen for further studies. The genes encoding PDC in these organisms were cloned and the pHCA decarboxylase expressed in Escherichia coli strains co-transformed with a plasmid encoding a bifunctional PAL/TAL enzyme from the yeast Rhodotorula glutinis. Production of pHS from glucose was ten-fold greater for the expressed L. plantarum pdc gene (0.11mM), compared to that obtained when the B. subtilis PDC gene (padC) was used. An E. coli strain (WWQ51.1) expressing both tyrosine ammonia-lyase(PAL) and pHCA decarboxylase (pdc), when grown in a 14L fermentor and under phosphate limited conditions, produced 0.4g/L of pHS from glucose. We, therefore, demonstrate pHS production from an inexpensive carbohydrate feedstock by fermentation using a novel metabolic pathway comprising genes from E. coli, L. plantarum and R. glutinis.     

Retroviral mediated transfer and expression of human alpha 1-antitrypsin in cultured cells

Genetic deficiency of alpha 1-antitrypsin in man is a predisposing factor to emphysema and a disorder potentially correctable by somatic gene therapy. A full-length human alpha 1-antitrypsin cDNA was cloned into a retroviral vector and introduced into cells which package the recombinant gene in a retroviral capsule. Cells infected with the recombinant retrovirus express human alpha 1-antitrypsin mRNA and protein. The recombinant protein is glycosylated, secreted and exhibits anti-protease activity against human neutrophil elastase.     

Construction of a neo fusion gene for expression in both prokaryotic and eukaryotic cells

A high-copy-number plasmid, pLink, was constructed to allow the direct selection in Escherichia coli of a neo fusion gene capable of conferring Geneticin (G418) resistance on mouse L cells. pLink was derived from pdMmtneo by insertion of a KpnI linker within the 5'-coding region of the neo gene. This created a minus-one frameshift mutation resulting in a translational termination within the N-terminal region of the protein. The Neo activity was restored by insertion into the modified neo gene of a piece of coding sequence derived from human HPRT cDNA. The resulting plasmid, pAH, was microinjected into mouse A9 cells and shown to confer resistance to G418.     

Pathways of arsenic uptake in prokaryotic and eukaryotic cells

Mechanisms of arsenic uptake and detoxification are present in all studied organisms. These mechanisms are considerably well described in unicellular organisms such as bacterium Escherichia coli and baker's yeast Saccharomyces cerevisiae, still leaving much to be revealed in multicellular organisms. Full identification of arsenic uptake and detoxification is of great importance. This knowledge can be very helpful in improving effectiveness of arsenic-containing drugs used in chemotherapy of parasitoses as well as in treatment of acute promielyocytic leukemia. Increased proficiency of bioremediation of arsenic-contaminated soils can be obtained by using plants hyperaccumulating arsenic. This kind of plants can be engineered by modulating expression levels of genes encoding arsenic transporters. The same technique may be used to decrease levels of accumulated arsenic in crops. The aim of this paper is to review current knowledge about systems of arsenic uptake in every studied organism--from bacteria to human.     

DNA-mediated transfer of cAMP resistance in CHO cells

Chinese hamster ovary (CHO) strain 10215 carries a dominant mutation which confers resistant to cAMP by virtue of an altered catalytic subunit of the cAMP-dependent protein kinase (Evain et al., 1979). This mutation was transferred to wild-type CHO cells by DNA-mediated gene transfer. Based on the absence of cAMP growth inhibition, seven transformant colonies were isolated. One of these, 11586, was studied in detail. This transformant showed the same phenotype as the mutant, including resistance to the morphological changes and growth inhibitory effects of 1 mM 8-Br-cAMP, reduced total cAMP dependent protein kinase activity and lowered sensitivity of the kinase to cAMP activation. When the cAMP-dependent protein kinase was fractionated on a DEAE-cellulose column, the transformant was lacking in type II cAMP dependent protein activity, to the same degree as the mutant. The transformant and mutant, but not wild-type cells, also failed to phosphorylate a 52,000-dalton protein in a cAMP-dependent manner. These characteristics support the conclusion that the gene for the mutant cAMP-dependent protein kinase has been transferred. The ability to transfer this gene by DNA-mediated transfer suggests that this methodology may be useful for the molecular isolation of the gene encoding the catalytic subunit of cAMP-dependent protein kinase.     

Comparative study of the expression of the native and modified cry3a genes of Bacillus thuringiensis in prokaryotic and eukaryotic cells

The cry3a gene of Bacillus thuriengiensis was cloned. Based on sequence analysis of this gene, a modified gene, cry3aM, was constructed, which has the optimal codon composition for effective expression in eukaryotic cells. Hybrid genes cry3a-licBM2 and cry3aM-licBM2 were constructed, in which the sequences of the native and modified genes are fusedfused with the reorter gene for thermostable lichenase in the reading frame. We have shown that the expression levels of hybrid genes cry3a-licBM2 and cry3aM-licBM2 in Escherichia coli are comparable, being 5% of those for reporter gene licBM2. In cells of a lower eukaryote Saccharomyces cerevisiae, the expression of hybrid gene cry3aM-licBM2? Which contains the modified gene, considerably exceeded the level of expression of cry3a-licBM2 containing the native gene. The presence of lichenase in the composition of hybrid proteins was shown to facilitate selection and analysis of the expression level of hybrid proteins in transgenic organisms.     

Mutation accumulation in nuclear, organelle, and prokaryotic transfer RNA genes

A comparative analysis of the transfer RNA genes in the genomes of the major kingdoms of eukaryotes and prokaryotes leads to the general conclusion that the rate of evolution of organelle tRNA genes is typically equal to of greater than that of their nuclear counterparts. Situations where this is not the case, most notably in vascular plants, are attributable to an elevated mutation rate in the nuclear genome. Through a comparison of rates of mutation with rates of nucleotide substitution, it is shown that there is a reduction in the efficiency of selection on new mutations in organelle genes. Numerous lines of evidence, including observed reductions in stem duplex stability and changes in loop sizes, suggest that the excess changes observed in the organelle genes are mildly deleterious. Uniparental inheritance of organelles causes a reduction in the efficiency of selection through the joint effects of an increase in linkage disequilibrium and a decrease in effective population size. These results provide molecular support for the idea that asexually propagating genomes are subject to long-term, gradual fitness loss and raise questions about the role of organelle mutations in the long-term survival of major phylogenetic lineages.      

A modular set of prokaryotic and eukaryotic expression vectors

A modular series of versatile expression vectors is described for improved affinity purification of recombinant fusion proteins. Special features of these vectors include (i) serial affinity tags (hexahistidine-GST) to yield extremely pure protein even with very low expression rates, (ii) highly efficient proteolytic cleavage of affinity tags under a variety of conditions by hexahistidine-tagged tobacco etch virus (TEV) protease, (iii) PCR cloning design that results in a product of proteolytic cleavage with only one (a single glycine) or two (gly-ala) amino acids at the N-terminus of the protein, and (iv) expression in either Escherichia coli or Saccharomyces cerevisiae. In addition, singly hexahistidine-tagged proteins can be produced for purification under denaturing conditions and some vectors allow addition of five amino acid kinase recognition sites for easy radiolabeling of proteins. To illustrate the use of these vectors, all regulatory components of the yeast GAL regulon, rather than abundant highly soluble proteins, were produced and purified under native or denaturing conditions, and their biological activity was confirmed.