Stringent regulation of human growth hormone expression in cultured murine C2C12 myoblasts by theE. coli lac repressor

Summary Gene transfer techniques can be used to encode the production of a polypeptide product, such as human growth hormone (hGH), that is missing in an acquired or inherited disease state such as growth hormone deficiency. In one model system, engineered C2C12 myoblasts are injected intramuscularly into a mouse and subsequently secrete hGH into the circulation. In this regard, a gene-expression regulatory system that functions in myoblasts would be of interest. We demonstrate that theEscherichia coli lac operon system can be used to stringently regulate the expression of hGH in engineered C2C12 myoblasts in tissue culture. A DNA segment encoding hGH was linked to a DNA segment containing an SV40 enhancer and promoter. The latter components were positioned between two syntheticlac operators.Lac repressor expression was driven by a simian cytomegalovirus promoter. In transient co-transfection assays, hGH expression from cultured C2C12 myoblasts could be modulated up to 60-fold (P = 0.002) with the inducing agent, isopropyl-β-d-thiogalactoside (IPTG). In the absence of IPTG, hGH expression was almost fully repressed. These results show that the components of theE. coli lac operon provide a stringent regulatory system for use in myoblasts. The system might prove to be useful for the regulation of transferred genes in animals.     

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.     

Operon fusions of Mu d1(Ap,lac) to thel-proline biosynthetic genes ofescherichia coli K-12

Operon fusions to the promoter of either theproA,proB, orproC genes of the proline biosynthetic pathway were obtained by the use of the Mu d1(Ap,lac) bacteriophage. These fusions were further stabilized by transformation with plasmid pGW600 containing the wildtype Mu repressor gene or by transduction with phage pSG1. The level of -galactosidase in the fusion strains was not affected by the presence of exogenously addedl-proline or high concentrations of NaCl in the growth medium. A Tn5 insertion nearproBA increased -galactosidase expression 140- to 200-fold in strains carrying theproA-lac andproB-lac fusions, but the level of this enzyme was unaltered in strains carrying theproC-lac fusion. The Tn5 insertion increased intracellular proline concentrations 8- to 10-fold, suggesting that mechanisms other than allosteric inhibition may regulate proline biosynthesis, but did not confer osmotolerance to cells growing in a medium with a high concentration of salt.     

Inducible protein expression in <Emphasis Type="Italic">Drosophila</Emphasis> Schneider 2 cells using the <Emphasis Type="Italic">lac</Emphasis> operator–repressor system

Schneider line 2 cells, derived from Drosophila melanogaster, can be used as a highly versatile gene expression system. Two powerful promoters derived from the actin5C (Ac5) and metallothionein (Mtn) genes are available. The Mtn promoter can be used for the inducible expression of heterologous proteins unsuitable for constitutive expression. However, to circumvent using CuSO₄ or CdCl₂ as inducers of the Mtn promoter, we created a modified Ac5 promoter, Ac5LacO, in which two short lac operator sequences are embedded. Expression from the Ac5LacO promoter was regulated with co-expression of the lac repressor and IPTG. More than 25-fold induction of firefly luciferase expression was achieved in transient transfection experiments. Furthermore, we demonstrated that the lac operator–repressor regulatory system functioned in chromosomally integrated cell lines.     

The possible roles of residues 79 and 80 of the Trp repressor from Escherichia coli K-12 in trp operator recognition

We constructed mutants of the Trp repressor from Escherichia coli K-12 with all possible single amino acid exchanges at positions 79 and 80 (residues 1 and 2 of the recognition helix). We tested these mutants in vivo by measuring the repression of synthesis of -galactosidase with symmetric variants of - and -centered trp operators, which replace the lac operator in a synthetic lac system. The Trp repressor carrying a substitution of isoleucine 79 by lysine, showed a marked specificity change with respect to base pair 7 of the -centered trp operator. Gel retardation experiments confirmed this result. Trp repressor mutant IR79 specifically recognizes a trp operator variant with substitutions in positions 7 and 8. Another mutant, with glycine in position 79, exhibited loss of contact at base pair 7. We speculate that the side chain of Ile79 interacts with the AT base pairs 7 and 8 of the -centered trp operator, possibly with the methyl groups of thymines. Replacement of thymine in position 7 or 8 by uracil confirms the involvement of the methyl group of thymine 8 in repressor binding. Several Trp repressor mutants in position 80 (i.e. AI80, AL80, AM80 and AP80) broaden the specificity of the Trp repressor for -centered trp operator variants with exchanges in positions 3, 4 and 5.     

Purification of Lac repressor protein using polymer displacement and immobilization of the protein

Lac repressor protein was purified from E. coli BMH8117 harboring plasmid pWB1000 and E. coli K₁₂BMH 71-18 strains. Displacement of the protein with poly(ethyleneimine) (PEI) from phosphocellulose cation exchange column was shown to be an effective elution strategy. It resulted in better recoveries and sharper elution profiles than traditional salt elution without effecting the purity of the protein. The elution is assumed to proceed via displacement of bound protein by PEI when the polymer binds to the ion exchanger. The minor impurities in the protein solution were finally removed by chromatography on immobilized metal affinity column. The repressor protein undergoes distinct conformational changes upon addition of specific inducer isopropyl--D-thiogalactoside (IPTG), which is evidenced by changes in ultraviolet absorption spectrum. The protein was immobilized covalently to the Sepharose matrix. The intact biological activity of the protein after immobilization was shown by binding of genomic DNA and lac operator plasmid DNA from E. coli to the immobilized lac repressor.     

Germline transmission of exogenous genes in chickens using helper-free ecotropic avian leukosis virus-based vectors

We have used vectors derived from avian leukosis viruses to transduce exogenous genes into early somatic stem cells of chicken embryos. The ecotropic helper cell line, Isolde, was used to generate stocks of NL-B vector carrying theNeo ^r selectable marker and theEscherichia coli lacZ gene. Microinjection of the NL-B vector directly beneath unincubated chicken embryo blastoderms resulted in infection of germline stem cells. One of the 16 male birds hatched (6.25%) from the injected embryos contained vector DNA sequences in its semen. Vector sequences were transmitted to G₁ progeny at a frequency of 2.7%.Neo ^r andlacZ genes were transcribedin vitro in chicken embryo fibroblast cultures from transgenic embryos of the G₂ progeny.     

Binding of lac repressor to the secondary lac operator in Escherichia coli.

Summary In the lac operon, the existence of a secondary repressor binding site, inside Z gene, had been inferred from in vitro binding studies (Reznikoff et al., 1974; Gilbert et al., 1975).A serie of deletions have been constructed from a lac transducing bacteriophage. Some of those deleted bacteriophages have still the property of derepressing a chromosomal lac operon, even though they do not contain any more the lac operator. This phenomenon is an indication that the secondary repressor binding site is also active in vivo.     

Evidence that the geneuvrB is indispensable for a polymerase I deficient strain ofEscherichia coli K-12

Summary The gene coding for bacteriophage Lambda repressor (cI gene) has been fused to the lac operon of Escherichia coli. In some of the fusions Lambda repressor synthesis can be controlled by the lac operator and promoter. Upon induction of the lac operon the amount of Lambda repressor is increased by a factor of 7 over that found in a single lysogen. In combination with the polarity suppressor suA the induction factor rises to 20. Transducing phages of one fusion were constructed. After thermal induction of this phage the final level of Lambda repressor was enhanced by a factor of 150.Abbreviations xgal 5-bromo-4-chloro-3-indolyl--D-galactoside - IPTG isopropyl-thio--D-galactoside     

Tetracycline operator/repressor system to visualize fluorescence-tagged T-DNAs in interphase nuclei ofArabidopsis

The bacterial tetracycline operator/repressor (tetO/TetR) system and enhanced yellow fluorescent protein (EYFP) have been adapted for use inArabidopsis to visualize tagged T-DNAs in interphase nuclei of living cells. The 2-component system was assembled on a single T-DNA construct that contained a gene encoding a nuclear-targeted TetR-EYFP fusion protein under the control of the 35S promoter of the cauliflower mosaic virus together with multiple tandemly arranged copies of thetetO. In a number of independentArabidopsis lines transformed with this construct, bright fluorescent dots corresponding to tagged T-DNAs were observed in interphase nuclei of various cell types using standard fluorescence microscopy. In selfed progeny of a single locus line, hemizygous and homozygous plants were distinguished by having 1 or 2 fluorescent dots, respectively. Low background fluorescence of EYFP in many plant tissues facilitates the visualization of tagged T-DNAs. We compared features of thetetO/TetR-EYFP system to a second system we developed on the basis of the bacteriallac operator/repressor and enhanced green fluorescent protein.     

Effect of thedpy-20 androl-6 cotransformation markers on α-tubulin gene expression inC. elegans transformants

An -1 tubulin::lacZ fusion gene was introduced into the germline ofCaenorhabditis elegans, using eitherrol-6 ordpy-20 genomic DNA as a cotransformation marker. Distinct patterns in cellular specificity of the -1 tubulin::lacZ fusion gene expression were observed, depending on the cotransformation marker used. For therol-6 marker, the tubulin fusion gene was expressed in several neurons in the head and tail ganglia and a set of 38–39 ventral cord motor neurons along the body length of the animal during larval and adult development. In contrast, for thedpy-20 marker system, not only were fewer neurons stained in the head and tail region, but also the staining of ventral cord motor neurons was extremely reduced both in number and intensity. Thedpy-20 marked-mediated suppression of the -1 tubulin gene expression was observed both in thecis andtrans configurations. Similar down-regulation in the ventral cord motor neurons was observed when the -2 tubulin::lacZ fusion gene construct was tested in these experiments using thedpy-20 marker. In controls, where the tubulin fusion gene was directly injected to obtain transformants without any marker DNA, the cellular staining pattern was close to the fusion gene expression observed with therol-6 marker DNA. These results underline the importance of the choice of transformation marker system in generation of the transgenic animals, and reveal a down-regulation of the -tubulin fusion gene expression in the ventral cord motor neurons in transgenic animals when thedpy-20 gene was used as a cotransformation marker.     

A tightly regulated expression system for <Emphasis Type="Italic">E. coli</Emphasis> using supersaturated silicic acid

Objective

To develop a new expression system regulated by a ferric uptake regulator in which silicic acid is used as an inducer.

Results

Fur box (binding site for Fur) was substituted for the lac operator to regulate the expression of GFP with the lac promoter. Since the addition of supersaturated silicic acid invokes iron deficiency, supersaturated silicic acids were used as an inducer. GFP expression was dependent on silica concentration, and the expression level without silica was negligible. Basal expression level of this lac-Fur system was extremely low and, hence, lytic enzyme gene E from bacteriophage ?X174 could be retained in this system. Furthermore, the expression of genes of interest was spontaneously initiated as the cell density increased and the costs of the inducer are considerably less than IPTG.

Conclusion

The combination of lac promoter and Ferric uptake repressor allowed the protein expression by supersaturated silicic acid as an inducer in an easy and cost-effective way.

     

Biosynthesis and secretion of recombinant human growth hormone in Pichia pastoris

Mature human growth hormone (hGH) cDNA was cloned by homologous recombination into the yeast Pichia pastoris genome. The hGH gene expression was placed under the control of the methanol-inducible alcohol oxidase 1 (AOX1) gene promoter and the Saccharomyces cerevisiae -factor signal sequence to direct the secretion of recombinant human growth hormone (rhGH) into the growth medium. O₂-limited induction of recombinant yeast strains in shake tubes with 3 ml of culture medium produced up to 11 mg rhGH l^–1, while high cell density cultures using a 2-l bioreactor produced about 49 mg rhGH l^–1 achieving 40% of total protein of the culture medium supernatant.     

Expression of c-fos and c-myc protooncogenes in an immortalized hepatocyte line harbouring SV40 T antigen and hGH as transgenes

A clonal hepatocyte line (FMH-202-2), derived from livers of fetal transgenic mice harbouring human growth hormone (hGH) and SV40 T antigen as transgenes, was used in the investigation of protooncogene expression involved in liver-specific growth control and/or in hepatocellular transformation. In this model system, representing an immortalized, yet untransformed phenotype, the transgenes hGH and SV40 T antigen were expressed constitutively. The c-fos protooncogene was induced by incubation with insulin, epidermal growth factor (EGF) and insulin-like growth factor (IGF-I) in a transient manner comparable to its expression in primary murine hepatocytes. Elucidation of second messenger mechanisms demonstrated that c-fos induction by hepatotrophic growth factors was not mediated by protein kinase C. In contrast to primary hepatocytes, the c-myc protooncogene exhibited a constitutive expression pattern which was independent of growth factor stimulation. These results indicate that apart from hGH and SV40 T antigen, c-myc may play a role in cellular immortalization, but that constitutive expression of these genes, even in combined coexpression, does not suffice to induce the transformed phenotype.     

Induction of the inhibitor of RecBCD enzyme inEscherichia coli is alexA-independent SOS response

Exonuclease V (ExoV), an enzyme involved in the RecBCD pathway of recombination, was inhibited in cells induced for SOS functions. In vitro experiments showed that an ExoV inhibitor (Exi) induced after SOS induction was responsible for the inhibition of ExoV. Unlike other SOS functions, Exi protein was induced even inlexA(Ind^–) mutants. Phage Mud(amp^r,lac) was fused to the promoter of theexi gene in alexA(Ind^–) strain, and in these fusion strains-galactosidase was inducible five- to six-fold after DNA damage. The Exi protein, in addition to the inhibition of ATP-dependent DNase activity of ExoV, appeared to repress the synthesis of polypeptide subunits of ExoV as well. Further, Exi protein appeared to be an inducible repressor of a number of other genes in SOS-induced cells.     

On the Mechanism of Action of <Emphasis Type="Italic">lac</Emphasis> Repressor

Chen et al. have proved conclusively that lac repressor and RNA polymerase bind independently to wild type lac DNA in vitro. To explain the lacp ^s mutation, which causes competitive binding between repressor and polymerase, they suggest that a new promoter site has been created near the lac operator.     

Glutamic acid and alanine spacer is not necessary for removal of MFα-1 signal sequence fused to the human growth hormone produced from Pichia pastoris

Human growth hormone (hGH) cDNA was synthesised using codons preferred by Escherichia coli, except for the first 20 amino acids, which were changed to that preferred by Saccharomyces cerevisiae and Pichia pastoris. Polymerase chain reaction (PCR) overlapping approach was employed to create synthetic hGH without glutamic acid-alanine (glu-ala), or with one and two glu-ala spacers (hGH, hGH1 and hGH2, respectively). The necessity of a glu-ala spacer in the cleavage of S. cerevisiae alpha mating factor-1 (MF-1) secretion signal from the synthetic hGH was also investigated. Three types of hGH constructs were integrated into P. pastoris genome, the zeocin-resistant transformants were selected and expression of hGH was determined. A 22-kDa band of secreted hGH was further determined by N-terminal peptide sequencing. The result suggested that the removal of glu-ala from the hGH1 and hGH2 was not efficient and only the hGH construct showed the complete cleavage of the signal sequence, giving a similar N-terminus as the mature hGH. hGH expression was optimized to increase the yield of the protein from the hGH construct (no glu-ala) to 190 mg/l from a 10-ml induction medium.