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PubMed1986

Stimulation in vitro of expression of the amp gene of pBR322 by soluble protein fractions isolated from E. coli.

Two soluble protein fractions isolated from E. coli were found to be required for efficient expression of the amp gene of pBR322 in an in vitro coupled transcription-translation system consisting of unwashed ribosomes and a polyethylene glycol-treated S30 extract from E. coli. Both fractions stimulated the pBR322-directed synthesis of the amp gene product, pre-beta-lactamase, in the in vitro system. However, pBR322-directed RNA synthesis was stimulated only by one fraction. It is therefore likely that one fraction enhances the transcription of the amp gene and the other is involved mainly in stimulation of the translation of pre-beta-lactamase mRNA.

Bacterial Proteins↗
PubMed1995

Expression and secretion of antifreeze peptides in the yeast Saccharomyces cerevisiae.

The antifreeze peptide AFP6 from the polar fish Pseudopleuronectus americanus has been expressed in and secreted by the yeast Saccharomyces cerevisiae as a biologically active molecule. The gene for the 37 amino acid long peptide has been chemically synthesized using yeast preferred codons. Subsequently, the gene has been cloned into an episomal expression vector as well as in a multicopy integration vector, which is mitotically more stable. The expression is under the control of the inducible GAL7 promoter. The enzyme alpha-galactosidase has been investigated as a carrier protein to facilitate expression and secretion of AFP. In order to reach increased expression levels, tandem repeats of the AFP gene (up to eight copies) have been cloned. In most cases the genes are efficiently expressed and the products secreted. The expression level amounts to approximately 100 mg/l in the culture medium. In a number of genetic constructs the genes are directly linked and expressed as AFP multimers. In other constructs linker regions have been inserted between the AFP gene copies, that allow the peptide to be processed by specific proteinases, either from the endogenous yeast proteolytic system or from a non-yeast source. The latter requires a separate processing step after yeast cultivation to obtain mature AFP. In all these cases proteolytic processing is incomplete, generating a heterogeneous mixture of mature AFP, carrier and chimeric protein, and/or a mixture of AFP-oligomers. The antifreeze activity has been demonstrated for such mixtures as well as for AFP multimers.

Amino Acid Sequence↗
PubMed1999

High-level expression of Rhizopus niveus lipase in the yeast Saccharomyces cerevisiae and structural properties of the expressed enzyme.

Rhizopus niveus lipase (RNL) has a unique structure consisting of two noncovalently bound polypeptides (A-chain and B-chain). To improve this enzyme's properties by protein engineering, we have developed a new expression system for the production of recombinant lipase in the yeast Saccharomyces cerevisiae. For the present study, we developed a more efficient expression system using the strain ND-12B and the multicopy-type plasmid pJDB219. We purified two types of recombinant lipases, each to a single peak by gel-filtration HPLC, although they were found to be heterogeneous by SDS-PAGE. Analysis of reversed-phase HPLC, N-terminal amino acid sequence, and sugar content showed that the difference between the two types of lipases was due mainly to their sugar content (high or low mannose type). Moreover, there were two species within each type of lipase. One kind was processed to the A-chain and B-chain as in the native lipase, while the other remained unprocessed. Although these yeast-purified lipases contained several posttranslational modifications and different glycosylations, their secondary structures were the same as those of the native lipase as measured by circular dichroism spectra and determination of disulfide bonding. This suggests that protein folding of the recombinant lipase occurred correctly in yeast.

Amino Acid Sequence↗
PubMed1991

High-level expression of enzymatically active bovine leukemia virus proteinase in E. coli.

An E. coli plasmid expressing efficiently an artificial precursor of bovine leukemia virus (BLV) proteinase under transcriptional control of the phage T7 promoter was constructed. The expression product accumulates in the induced E. coli cells in the form of insoluble cytoplasmic inclusions. Solubilization of the inclusions and a refolding step yield almost pure and completely self-processed proteinase. Purification to homogeneity was achieved by ion-exchange chromatography and reverse-phase HPLC. On a preparative scale, a high yield of enzymatically active proteinase was obtained. An initial study using a series of synthetic peptide substrates shows a distinct substrate specificity of BLV proteinase.

Amino Acid Sequence↗
PubMed1985

Differential effect of poly rI.rC and Newcastle disease virus on the expression of interferon and cellular genes in mouse cells.

The expression of type I murine interferon (MuIFN) genes and several other cellular genes was examined in poly rI.rC induced and Newcastle disease virus (NDV) infected mouse cells. Northern analysis of RNA from induced L cells revealed that the MuIFN-alpha s are expressed efficiently in NDV infected cells but only at low levels in poly rI.rC induced cells. MuIFN-beta 1, however, is expressed equally well in cells treated with poly rI.rC or infected with NDV. As shown by the use of a probe specific for poly rI.rC, interferon induction correlates with the cellular uptake of poly rI.rC into the cells. The relative levels of alpha and beta 1 mRNAs in the cells reached a maximum at 10 hr after the induction which indicates coordinate expression of alpha and beta 1 interferon genes. The effect of viral infection on the expression of two murine genes coinduced with interferon (pMIF20/11 and pMIF3/10) and several cellular genes was also examined. While pMIF20/11 is an inducible gene, the pMIF3/10 gene is expressed constitutively in mouse L cells. Viral infection, but not poly rI.rC treatment, enhanced the expression of the pMIF3/10 gene, as well as two other cellular genes; H-2 and c-myc, however, the expression of beta-actin gene was unaltered. These data indicate that enhancement of gene expression in virus infected cells in not limited to the interferon system.

Actins↗
PubMed1993

Construction and expression of a synthetic streptavidin-encoding gene in Escherichia coli.

A synthetic gene encoding 'core' streptavidin (SAV) [amino acid (aa) residues 13-140 of Streptomyces avidinii SAV] has been efficiently expressed in Escherichia coli from the IPTG-inducible lac promoter of plasmid pET3a. In this system, expression levels are nearly tenfold greater for the synthetic gene than for the corresponding native gene. The synthetic gene was constructed from overlapping oligodeoxyribonucleotides whose sequences were optimized to incorporate codons preferred by highly expressed E. coli genes. Biochemical characterizations by gel methods, aa analysis, N-terminal sequencing, and size exclusion chromatography show that the synthetic gene product purified by affinity chromatography possesses the properties expected for core SAV.

Amino Acid Sequence↗
PubMed1999

High-level expression, purification, and some properties of a recombinant cephalosporin-C deacetylase.

To maximize the expression of the cephalosporin-C deacetylase (CAH) gene isolated from Bacillus subtilis SHS 0133 in Escherichia coli, a series of expression plasmids was constructed with various spacings between the Shine-Dalgarno sequence and the ATG initiation codon. As the most efficient expression plasmid, we selected pCAH431, which has the trp promoter, a replication origin derived from pAT153, and a spacing of 13 nucleotides. E. coli JM103 with pCAH431 produced 4.9 g of CAH per liter on cultivation at 37 degrees C for 20 h in a 30-l jar fermentor. Since the amount of CAH reached about 70% of the total protein in the soluble fraction of the cells, and CAH was recovered from the cell extracts in an active form, the CAH was purified easily to homogeneity by only one column chromatography step. Twenty grams of 7-aminocephalosporanic acid was completely converted to deacetyl-7-aminocephalosporanic acid, a starting material for cefcapene pivoxil hydrochloride, by 12 mg of the purified enzyme without significant appearance of by-products. Thus, our expression and purification system has made the industrial production of CAH possible.

Journal Article↗
PubMed2002

Size and topology of exogenous DNA as determinant factors of transgene transcription in mammalian cells.

We prepared a series of linearized DNA duplexes of various lengths in order to examine the effects of topology and the size of exogenous, plasmid-derived DNAs on transgene expression. These linearized DNA duplexes were capped at each end with a highly stable loop (5'-GCGAAGC-3') to produce a dumbbell-shaped construct that is refractory to exonuclease digestion in comparison to the analogous uncapped DNA duplexes. Intranuclear microinjection of the DNA dumbbells into simian COS-7 cells allowed the expression of the green fluorescent protein (GFP) gene on the linearized molecules, which was expressed five- to 10-fold more than that on the circular DNA of the same size. In addition, the expression by the dumbbell DNA was higher than that by the circular plasmid for at least 14 days. Interestingly, the size of the dumbbell DNA affected the transgene expression upon their microinjection into cell nuclei. The GFP expression efficiency increased with decreasing DNA size below a DNA size of 5.7 kb. The effects of topology and size on the expression of DNAs transfected with cationic lipids are similar to those of DNAs microinjected into cell nuclei. In contrast, microinjection into the cytosol showed the inverse size dependency over a range of 2.3 to 9.4 kb. Thus, transcription of a transgene in the nucleus, but not endocytosis or nuclear entry, was influenced by the exogenous DNA structure, and this was the primary determinant of transgene expression upon transfection under our conditions. These results indicate that small, linearized DNA duplexes that are end-capped with a highly stable loop (dumbbell-shaped DNA) would be very useful for nonviral gene therapy.

3T3 Cells↗
PubMed2000

Transcriptional activation of the human hematopoietic prostaglandin D synthase gene in megakaryoblastic cells. Roles of the oct-1 element in the 5'-flanking region and the AP-2 element in the untranslated exon 1.

The human hematopoietic prostaglandin D synthase (H-PGDS) gene is highly expressed in human megakaryoblastic cells, in which phorbol ester induces its expression. We characterized the promoter activity of the 5'-flanking region and the untranslated exon 1 (-1044 to +290) of the human H-PGDS gene in human megakaryoblastic Dami cells. Transient expression analysis using the luciferase reporter gene revealed that the 5'-flanking region and the untranslated exon 1 were sufficient for efficient expression of the H-PGDS gene in Dami cells, but not in monocytic U937 cells. Deletion and site-directed mutagenesis of the Oct-1 element in the 5'-flanking region decreased the promoter activity by approximately 30% compared with that of the entire region from -1044 to +290. An electrophoretic mobility shift assay demonstrated that Oct-1 specifically bound to the promoter region. Interestingly, even only untranslated exon 1 (+1 to +290) showed approximately 60% of the promoter activity of the entire region from -1044 to +290. Site-directed mutagenesis of the AP-2 element within the untranslated exon 1 abolished the basal promoter activity as well as its phorbol ester-mediated up-regulation. In AP-2-deficient HepG2 cells, the H-PGDS promoter activity was enhanced by coexpression with AP-2alpha. These findings indicate that the Oct-1 element in the 5'-flanking region acts as a positive cis-acting element and that the AP-2 element in the untranslated exon 1 is crucial for both basal and phorbol ester-mediated up-regulation of human H-PGDS gene expression in megakaryoblastic Dami cells.

Base Sequence↗
PubMed1993

Ectopic synthesis of epidermal cytokeratins in pancreatic islet cells of transgenic mice interferes with cytoskeletal order and insulin production.

The members of the multigene family of intermediate filament (IF) proteins are expressed in various combinations and amounts that are specific for a given pathway or state of differentiation. Previous experiments in which the cell type-specific IF cytoskeleton was altered by introducing foreign IF proteins into cultured cells or certain tissues of transgenic animals have shown a remarkable tolerance, without detectable interference with cell functions. To examine the importance of the cell type-specific cytokeratin (CK) IF pattern, we have studied the ectopic expression of CK genes in different epithelia of transgenic mice. Here we report changes observed in the beta cells of pancreatic islets expressing the genes for human epidermal CKs 1 and/or 10 brought under control of the rat insulin promoter. Both genes were efficiently expressed, resulting in the appearance of numerous and massive bundles of aggregated IFs, resembling those of epidermal keratinocytes. While the synthesis of epidermal CK 10 was readily accommodated and compatible with cell function, mice expressing CK 1 in their beta cells, alone or in combination with CK 10, developed a special form of diabetes characterized by a drastic reduction of insulin-secretory vesicles and of insulin-and CK 1-producing cells. In many CK 1-producing cells, accumulations of fibrous or granular material containing CK 1 were also seen in the nucleus. This demonstration of functional importance of the specific CK-complement in an epithelial cell indicates a contribution of cell type-specific factors to cytoplasmic IF compartmentalization and that the specific CK complement can be crucial for functions and longevity of a given kind of epithelium.

3T3 Cells↗
PubMed1986

Cloning and expression in Escherichia coli of a synthetic DNA for hirudin, the blood coagulation inhibitor in the leech.

A 235-bp DNA coding for the leech blood coagulation inhibitor, hirudin, was chemically synthesized. The synthesis involved preparation of seven long oligodeoxyribonucleotide pairs which were assembled and cloned using a rapid and simple procedure. More than half of the transformed Escherichia coli cells expressed a biosynthetic polypeptide having biological properties which were very similar to authentic hirudin from the leech Hirudo medicinalis. To achieve efficient expression, we fused the hirudin DNA to a truncated C1 repressor gene of bacteriophage lambda to create a hybrid protein. An additional methionine at the fusion point allowed the active hirudin to be cleaved off by cyanogen bromide.

Animals↗
PubMed2002

Macromolecular uptake is a spontaneous event during mitosis in cultured fibroblasts: implications for vector-dependent plasmid transfection.

The process through which macromolecules penetrate the plasma membrane of mammalian cells remains poorly defined. We have examined whether natural cellular events modulate the capacity of cells to take up agents applied extraneously. Herein, we report that during mitosis and in a cell type-independent manner, cells exhibit a natural ability to absorb agents present in the extracellular environment up to 150 kDa as assessed using fluorescein isothiocyanate-dextrans. This event is exclusive to the mitotic period and not observed during G0, G1, S, or G2 phase. During mitosis, starting in advanced prophase, oligonucleotides, active enzymes, and polypeptides are efficiently taken into mitotic cells. This uptake of macromolecules during mitosis still takes place in the presence of cytochalasin D or nocodazole, showing no requirement for intact microtubules or actin filaments in this process. However, cell rounding up, which still takes place in the presence of either of these drugs in mitotic cells, appears to be a key event in this process. Indeed, limited trypsinization of adherent cells mimics both the cell retraction and macromolecule uptake observed as cells enter mitosis. A plasmid DNA encoding green fluorescent protein (3.3Mda) coated with an 18 amino acid peptide is efficiently expressed when applied onto synchronized G2/M fibroblasts, whereas little or no expression is observed when the coated plasmid is applied onto asynchronous cell cultures. This shows that such coating peptides are only efficient for their encapsulating and protective effect on the plasmid DNA to be "vectorized" rather than acting as true vectors.

Animals↗
PubMed1992

Transactivation of geminivirus AR1 and BR1 gene expression by the viral AL2 gene product occurs at the level of transcription.

Tomato golden mosaic virus is a bipartite geminivirus whose genome is divided between two circular DNA molecules. DNA A encodes functions necessary for viral DNA replication and encapsidation, whereas DNA B provides functions needed for movement in the host. Previous studies have shown that the viral AL2 gene product transactivates expression of the coat protein gene (AR1). We have investigated the role of the AL2 protein in the regulation of B component gene expression and examined the transcriptional and post-transcriptional components of this regulation. We found that AL2 protein is required for efficient expression of both the AR1 and BR1 genes, but not the BL1 gene. A comparison of steady state transcript levels and transcript levels determined by nuclear run-on analysis showed that activation of AR1 and BR1 gene expression by the AL2 protein occurs primarily at the level of transcription. These results provide an explanation for the lack of infectivity demonstrated by AL2 mutants, and suggest that the AL2 protein interacts with the cellular transcription machinery to activate the expression of rightward viral genes.

Amino Acid Sequence↗
PubMed1995

An internal ribosomal entry signal in the rat VL30 region of the Harvey murine sarcoma virus leader and its use in dicistronic retroviral vectors.

The genetic organization of the 5' genomic RNA domain of the highly oncogenic Harvey murine sarcoma virus appears to be unusual in that a multifunctional untranslated leader precedes the v-ras oncogene. This 5' leader is 1,076 nucleotides in length and is formed of independent regions involved in key steps of the viral life cycle: (i) the Moloney murine leukemia virus 5' repeat, untranslated 5' region, and primer binding site sequences necessary for the first steps of proviral DNA synthesis, (ii) the virus-like 30S (VL30)-derived sequence containing a functional dimerization-packaging signal (E/DLS) directing viral RNA dimerization and packaging into MLV virions, and (iii) an Alu-like sequence preceding the 5' untranslated sequence of v-rasH which contains the initiation codon of the p21ras oncoprotein. These functional features, the unusual length of this leader (1,076 nucleotides), and the presence of stable secondary structures between the cap and the v-ras initiation codon might well cause a premature stop of the scanning ribosomes and thus inhibit v-ras translation. In order to understand how Harvey murine sarcoma virus achieves a high level of expression of the ras oncogene, we asked whether the rat VL30 sequence, 5' to v-ras, could contribute to an efficient synthesis of the ras oncoprotein. The implications of the VL30 sequence in the translation initiation of Ha-ras were investigated in the rabbit reticulocyte lysate system and in murine cells. Results show that the rat VL30 sequence allows a cap-independent translation of a downstream reporter gene both in vitro and in murine cells. Additional experiments performed with dicistronic neo.VL30.lacZ mRNAs indicate that the 5' VL30 sequence (positions 380 to 794) contains an internal ribosomal entry signal. This finding led us to construct a new dicistronic retroviral vector with which the rat VL30 sequence was able to direct the efficient expression of a 3' cistron and packaging of recombinant dicistronic RNA into murine leukemia virus virions.

3T3 Cells↗
PubMed1994

Targeted expression of the E6 and E7 oncogenes of human papillomavirus type 16 in the epidermis of transgenic mice elicits generalized epidermal hyperplasia involving autocrine factors.

The E6 and E7 early genes of human papillomavirus type 16 have been shown in vitro to play a central role in the transforming capability of this virus. To explore their effects on differentiating epithelial cells in vivo, we used a bovine cytokeratin 10 (K10) promoter to target the expression of E6 and E7 to the suprabasal layers of the epidermis of transgenic mice. In two different lines of mice efficiently expressing the transgene, animals displayed generalized epidermal hyperplasia, hyperkeratosis and parakeratosis in the skin and the forestomach, both known to be sites of K10 expression. Northern (RNA) blot analysis revealed high levels of E6 and E7 transcripts, and in situ hybridizations localized these transcripts to the suprabasal strata of epidermis. In vivo labeling of proliferating cells showed two distinct effects of E6 and E7 expression in the epidermis: (i) an increase in the number of growing cells in the undifferentiated basal layer and (ii) abnormal proliferation of differentiated cells in the suprabasal strata. The expression of c-myc in the skin of transgenics was higher than that in control animals. The induction of c-myc transcription by topical application of tetradecanoyl phorbol acetate was prevented by simultaneous treatment with transforming growth factor beta 1 in nontransgenic skin but not in transgenic skin. In addition, transforming growth factor alpha was found to be overexpressed in the suprabasal layers of the transgenic epidermis. These findings suggest that autocrine mechanisms are involved in the development and maintenance of epidermal hyperplasia. Animals of both lines developed papillomas in skin sites exposed to mechanical irritation and wounding, suggesting that secondary events are necessary for progression to neoplasia. Collectively, these results provide new insights into the tumor promoter activities of human papillomavirus type 16 in epithelial cells in vivo.

Animals↗
PubMed1992

Recombinant mouse prolactin: expression in Escherichia coli, purification and biological activity.

Transformation of Escherichia coli cells with a recombinant plasmid containing modified mouse prolactin (mPRL) cDNA and a pKK223-3 vector resulted in efficient expression of mPRL protein. Cloned mPRL cDNA was modified by removing the 5' non-translating sequence as well as the sequence which encoded the signal peptide of preprolactin for recombination. In addition, approximately 100 nucleotides of the 5'-terminal region of the cDNA, which include the ATG initiation codon and the following 31 codons of mature mPRL, were replaced by a chemically synthesized oligonucleotide duplex. The sequence of this duplex was chosen to be rich in AT without changing the amino acid sequence of the protein. The modified cDNA was finally inserted into the multicopy plasmid, pUC19, before high-level expression of mPRL in E. coli cells was obtained. Western blotting analysis of total protein from transformed E. coli cells showed that both 23 and 16 kDa peptides were recognized by specific mPRL antisera. The purified and refolded 23 kDa protein exhibited a growth-stimulating effect on rat Nb 2 Node lymphoma cells, and was very similar to that of natural pituitary PRL.

Amino Acid Sequence↗
PubMed2001

Genetic modification of hematopoietic progenitor cells for combined resistance to 4-hydroperoxycyclophosphamide, vincristine, and daunorubicin.

AIM: To investigate whether human peripheral blood hematopoietic progenitor cells (PBPC) modified with human aldehyde dehydrogenase class-3 gene (ALDH-3) and multidrug resistance gene 1 (MDR1) would increase chemotherapy resistance to 4-hydroperoxycyclophosphamide (4-HC) and -glycoprotein effluxed drugs. METHODS: A bicistronic retroviral vector G1Na-ALDH3-IRES-MDR1 cDNA was constructed and used to transfect the packaging cell lines PA317 by electroporation. CD34+ PBPC were isolated with a high-gradient magnetic cell sorting system (MACS), and then were transfected with supernatant of retrovirus containing human ALDH-3 and MDR1 cDNA. PCR, RT-PCR, Southern blot, Northern blot, FACS, and MTT assay were used to evaluate the transfection and expression of the transgene in target cells. RESULTS: The bicistronic retroviral vector construction was verified by PCR and restriction endonuclease analysis. Dual drug resistance genes were integrated into the genomic DNA of CD34+ PBPC and expressed efficiently. The efficiency of gene transfection in CD34+ PBPC was tested to be 18 % on colonies. Nested PCR and Neor rescue assay indicated that no helper virus was present in this system. Compared with the untransduced cells, transgene recipient cells conferred 4.5-fold resistance to 4-HC, 6.6-fold and 7.8-fold resistance to P-glycoprotein effluxed drug, vincristine and daunorubicin, respectively. CONCLUSION: Efficient transduction of two different types of drug resistance genes into human peripheral blood hematopoietic progenitor cells and the co-expression may decrease cumulative myelosuppression of combination chemotherapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

[Effects of aldose reductase on the expression of fibronectin and collagen IV in cultured rat renal mesangial cells].

OBJECTIVE: To study the effect of aldose reductase (AR) on expression of fibronectin and collagen IV in cultured rat renal mesangial cells (MsC). METHODS: AR expression plasmid vector (pCDNA3-AR) was constructed by restriction endonuclease digestion and ligation procedures. Stable expression of AR in MsC was established by Lipofectin transfection. Western blot and immunofluorescence analyses were performed to verify the transfection efficiency. Expression of fibronectin and collagen IV proteins were analyzed using Western blot. RESULTS: Expression of fibronectin and collagen IV in naive MsC treated with TGF-beta1 was upregulated in comparison to that of the untreated naive MsC (P < 0.01). MsC transfected with pCDNA3-AR showed a remarkable increase of expression of fibronectin and collagen IV (P < 0.01). Aldose reductase inhibitors (Sorbinil and Zopolrestat) significantly inhibited the expression of fibronectin and collagen IV in naive MsC (P < 0.05). CONCLUSIONS: Overexpression or inhibition of AR activity significantly alters the expression of fibronectin and collagen IV proteins in cultured rat MsC, suggesting that AR plays a significant role in the pathogenesis of glomerulosclersis.

Aldehyde Reductase↗