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PubMed1987

Translation products of cauliflower mosaic virus ORF V, the coding region corresponding to the retrovirus pol gene.

Open reading frame (ORF) V of cauliflower mosaic virus (CaMV), the candidate for the reverse transcriptase gene, has been expressed in E. coli under control of the PR promoter of bacteriophage lambda either as an N-terminal polypeptide fused to beta-galactosidase or as the total ORF V without fusion. Antibodies against these proteins were used to analyze extracts from CaMV-infected plants by immunoblotting. ORF V-specific polypeptides of 80, 62, 58, 22, and 18 kD apparent molecular weights were detected, with the largest species corresponding to the full length translation product. The 62 and 22 kD species could be assigned to the N-terminus and the remaining two species to the C-terminus of the ORF.

Bacteriophage lambda↗
PubMed1993

Biologically active human and mouse nerve growth factors secreted by the yeast Saccharomyces cerevisiae.

Nerve growth factor (NGF) is a trophic agent that is essential for the development and survival of sympathetic and sensory nerves. A chemically-synthesized DNA fragment encoding human NGF (hNGF) and a cDNA encoding mouse NGF (mNGF) were engineered for expression in the yeast, Saccharomyces cerevisiae. Expression and secretion of hNGF and mNGF was attempted under the direction of the yeast PGK promoter and with various leader sequences. Among the leader sequences tested, that of the yeast alpha-factor successfully directed secretion of both hNGF and mNGF that were correctly processed. The content of the recombinant NGF (reNGF) in the culture supernatant was estimated to be 1 microgram/ml. The yeast-produced reNGF was able to bind to NGF receptors in rat pheochromocytoma (PC12) cells as efficiently as the standard mNGF, and partially purified reNGF could induce neurite outgrowth of PC12 cells. Thus, we have demonstrated that biologically active human and mouse reNGF can be produced in yeast cells.

Amino Acid Sequence↗
PubMed1980

On procaryotic gene expression in eucaryotic systems.

Numerous types of interaction between pro- and eucaryotes exist in nature, from the endosymbiosis of some bacteria with unicellular organisms and insects to the complex systems of bacterial flora associated with the skin and intestines of animals and man, and nitrogen-fixation and crown-gall tumor induction in plants. Until recently, such interactions were not thought to include genetic transfer, but an increasing body of evidence points to the probability of similar naturally-occurring exchanges with wide-ranging implications for evolution and genetic manipulation. Experiments to elucidate the possible effects of procaryotic genes in eucaryotic systems have included in vitro and in vivo studies with both plant and animal systems, for instance the translation of bacterial messenger RNAs in the wheat germ and rabbit reticulocyte systems and the introduction of bacterial genes into plant protoplasts, animal cells and whole organisms. In the present paper we have tried to summarize the results of experiments involving the uptake, replication, transcription, translation and integration of procaryotic genes in various eucaryotic systems and to discuss the implications of such findings for basic research as well as for possible biomedical applications. Awareness of the possibility of procaryotic-eucaryotic genetic interactions may help to elucidate unresolved questions in pathology, such as possible involvement of the intestinal flora in carcinogenesis, as well as to provide valuable probes of eucaryotic control mechanisms and new approaches in agricultural genetic engineering.

Animals↗
PubMed1986

Molecular basis of alpha 1-antitrypsin deficiency and its potential therapy by gene transfer.

The gene for alpha 1-antitrypsin, a serum anti-protease, has been cloned and sequenced. The underlying mutation in the PiZ allele has been identified as a G to A conversion giving rise to the substitution of glu by lys at position 342. Preparation of specific probes has allowed prenatal diagnosis. Recombinant retroviruses containing the normal human alpha 1-antitrypsin gene have been constructed and used to infect NIH3T3 cells. Analysis of DNA, RNA and protein indicate that successful incorporation of the alpha 1-antitrypsin was achieved and that the gene was capable of being expressed. The feasibility of genetic replacement therapy has been demonstrated and further experiments justified.

Base Sequence↗
PubMed1986

Direct alteration of a gene in the human genome.

Direct alteration of a gene in the human genome requires an understanding of the role of the gene in metabolism. A gene may need to be introduced into a specific tissue or alternatively it may be possible to use accessible tissue such as bone marrow. The level of gene expression required also needs to be known as does the position in the genome into which the gene is to be inserted. Insertion of DNA needs to be of high efficiency and accuracy. Various methods are available including virus, the use of inert adjuvant, microinjection and electroporation. The procedure with the most potential for accuracy is the use of specially designed plasmids. The example of the use of such a plasmid in achieving target modification of the beta-globin gene is given. The method has high accuracy but low efficiency.

DNA↗
PubMed2002

An insight into the possible mechanism of working of two-cistronic gene expression systems and rational designing of newer systems.

The initial attempts at hyper-expressing buffalo/goat growth hormone (GH)-ORFs in Escherichia coli directly under various strong promoters were not successful despite the presence of a functional gene. High level expression of GH was achieved as a fusion protein with glutathione-S-transferase (GST). To produce native GH in an unfused state, we adapted an established strategy of two-cistronic approach in our system. In this strategy, utilizing one of the highly efficient reported sequences as the first cistron led to a nearly 1000-fold enhancement in the level of expression under an E. coli promoter (trc). In search of a newer first-cistron sequence as well as to see the generality of the two-cistronic approach, we explored the ability of different lengths of a highly expressing natural gene to act as an efficient first cistron. Surprisingly, GST, which is naturally highly expressible in E. coli, could not be fitted into a successful two-cistronic construct. In addition, placement of the entire two-cistronic expression cassette (which had earlier given high-level GH expression under trc promoter) under the T7 promoter in E. coli failed to hyper-express GH. These results suggest that the successful exploitation of the two-cistron arrangement for hyper-expression of eukaryotic ORFs in bacteria is not as straightforward as was previously thought. It appears probable that factors such as the sequence context, together with the length and codons used in the first cistron are important as well.

Amino Acid Sequence↗
PubMed2001

Study on the preparation of recombinant human HSP70 and its presenting-antigen function.

The preparation of recombinant human HSP70 and its presenting-antigen function were investigated. Cultured in glucose-free M9ZB medium and induced with IPTG and lactose at a final concentration of 0.02 mmol/L and 5 mmol/L respectively, the engineered bacteria carrying expression vector of human HSP70 gene expressed rHSP70 at an efficiency fo 60%. After the purification with DEAE ion-exchange chromatography, HSP70 with a purity of higher than 90% was obtained. The purified product could bind tumor-antigen peptide in vitro, and the binding was identified by native PAGE containing 5% glycerol. HSP70-peptide complex could activate lymphocytes to produce specific cytotoxicity to tumor cells, suggesting that the recombinant human HSP70 could be used as an antigen-presenting reagent in tumor therapy.

Animals↗
PubMed2003

Non-conventional yeasts as producers of polyhydroxyalkanoates--genetic engineering of Arxula adeninivorans.

The non-conventional yeast Arxula adeninivorans was equipped with the genes phbA, phbB and phbC of the polyhydroxyalkanoate (PHA) biosynthetic pathway of Ralstonia eutropha, which encode beta-ketothiolase, NADPH-linked acetoacetyl-CoA reductase and PHA synthase, respectively. Arxula strains transformed solely with the PHA synthase gene (phbC) were able to produce PHA. However, the maximum content of the polymer detected in these strains was just 0.003% poly-3-hydroxybutyrate (PHB) and 0.112% poly-3-hydroxyvalerate (PHV). The expression of all three genes (phbA, phbB, phbC) resulted in small increases in the PHA content of the transgenic Arxula cells. However, under controlled cultivation conditions with minimal medium and ethanol as the carbon source, the recombinant yeast was able to accumulate up to 2.2% PHV and 0.019% PHB. Possible reasons for these differences are discussed.

Acetyl-CoA C-Acyltransferase↗
PubMed2005

The solvent-tolerant Pseudomonas putida S12 as host for the production of cinnamic acid from glucose.

A Pseudomonas putida S12 strain was constructed that efficiently produced the fine chemical cinnamic acid from glucose or glycerol via the central metabolite phenylalanine. The gene encoding phenylalanine ammonia lyase from the yeast Rhodosporidium toruloides was introduced. Phenylalanine availability was the main bottleneck in cinnamic acid production, which could not be overcome by the overexpressing enzymes of the phenylalanine biosynthesis pathway. A successful approach in abolishing this limitation was the generation of a bank of random mutants and selection on the toxic phenylalanine anti-metabolite m-fluoro-phenylalanine. Following high-throughput screening, a mutant strain was obtained that, under optimised culture conditions, accumulated over 5 mM of cinnamic acid with a yield (Cmol%) of 6.7%.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed2001

Cell surface-engineered yeast displaying a histidine oligopeptide (hexa-His) has enhanced adsorption of and tolerance to heavy metal ions.

A histidine oligopeptide (hexa-His) with the ability to chelate divalent heavy metal ions was displayed on the yeast cell surface for the purpose of enhanced adsorption of heavy metal ions. We genetically fused a hexa-His-encoding gene with the gene encoding the C-terminal half of alpha-agglutinin that includes a glycosylphosphatidylinositol anchor attachment signal sequence and attached the hexa-His peptide on the cell wall of Saccharomyces cerevisiae. This surface-engineered yeast adsorbed three to eight times more copper ions than the parent strain and was more resistant to copper (4 mM) than the parent (below 1 mM at pH 7.8). It was possible to recover about a half of the copper ions adsorbed by whole cells with EDTA treatment without disintegrating the cells. Thus, we succeeded in constructing a novel yeast cell with both tolerance to toxic contaminants and enhanced adsorption of metal ions onto the cell surface.

Adsorption↗
PubMed1997

The presence of complete but masked freezing nuclei in various artificially constructed ice nucleation-active proteobacteria.

Disparate gamma-subdivision proteobacteria artificially endowed with the same ice gene of enteric origin acquired water-freezing potential at -12 degrees C, but expressed it to varying extents under identical conditions of culture as well as after being subjected to certain post-culture treatments. Varying rates of cell-bound ice nucleus synthesis were probably not the root cause of these observed interspecies differences in nucleation-active cell frequency because potentially functional but masked ice-forming templates were found in the outer cell envelope of even initially inactive individuals taken from physiologically uniform populations of virtually all tested species. We therefore propose that the extent of bacterial ice nucleation generally reflects species-specified extent of ice nucleus sequestration.

Anti-Bacterial Agents↗
PubMed2004

A new selection method for pepper transformation: callus-mediated shoot formation.

We used two genes, TMV-CP and PPI1 (pepper-PMMV interaction 1 transcription factor), to transform commercially important chili pepper (Capsicum annuum) inbred lines (P915, P409) by means of Agrobacterium co-culture. Eighteen independently transformed T0 plants were obtained. The most critical point in the pepper transformation protocol was the selection of shoots growing on calli--referred to as callus-mediated shoot formation (indirect shooting)--because shoots not grown from the callus (direct shooting from the wounded surface) developed into non-transformants. Selection of the correct right callus type also proved to be an important requirement for obtaining transformed peppers. Six different types of callus developed during the selection process. Shoots regenerated from two of these types, while one type regenerated significantly more shoots than the other types, suggesting that the capacity for shoot formation is callus type-specific. Although the transformation rate was low, transformation via callus-mediated shoot formation proved to be reproducible and was confirmed by Southern and Northern blot analyses. Based on the experimental data, we have succeeded in developing a new protocol for the selection and transformation of pepper and expect that it will be used in the future for pepper transformation.

Capsicum↗
PubMed2004

Agrobacterium tumefaciens-mediated transformation of Campanula carpatica: factors affecting transformation and regeneration of transgenic shoots.

An efficient transformation system for Campanula carpatica was developed using Agrobacterium tumefaciens strains LBA4404 (harbouring the plasmid pBI121), and AGL0 (harbouring the plasmid pBEO210). This is the first report on the transformation of C. carpatica. Various factors affecting the transformation efficiency and subsequent regeneration were identified. The age of seedlings from which the explants for transformation studies were taken, and the growth conditions under which the seedlings were grown had a significant influence on the production of transformed shoots. Hypocotyls taken from 12-day-old seedlings grown in the dark were the most productive, with up to 25% of hypocotyls producing transformed shoots. Explants taken from 5-week-old seedlings produced only transformed callus. The medium used for co-cultivation and incubation also had a significant influence on transformation frequency and shoot regeneration. The cultivar "Blue Uniform" was more responsive than "White Uniform". Both bacterial strains and plasmids were equally effective in producing transformed tissue. Transformed shoots were selected on kanamycin medium, and the presence of the uidA and nptII genes in those selected shoots was confirmed by beta-glucuronidase and ELISA analyses, respectively.

Agrobacterium tumefaciens↗
PubMed2004

Growth and terpenoid indole alkaloid production in Catharanthus roseus hairy root clones in relation to left- and right-termini-linked Ri T-DNA gene integration.

Hairy root cultures of Catharanthus roseus var. Prabal were established by infecting the leaves with Agrobacterium rhizogenes agropine-type A4 strain. Two hundred and fifty independent root clones were evaluated for growth, morphology, number of integration of Ri T-DNA genes and alkaloid contents. On the basis of growth pattern, type of branching and number of lateral roots we were able to separate the hairy root clones into four categories. However based on the integration of the Ri T(L)-DNA and T(R)-DNA genes, there were only three different categories of independent hairy root clones-C1 (rolA&B(+)/ags(+)), C2 (rolA&B(-)/ags(+)) and C3 (rolA&B(+)/ags(-)). Southern hybridization analysis revealed both single and multiple copies of T-DNA integration in the root clones. The accumulation of considerable amounts of the root-specific alkaloids ajmalicine and serpentine was observed in the presence of both the T(L)-DNA and T(R)-DNA genes (C1) and the T(L)-DNA gene (C3) alone. Two rolA&B(-) but ags(+) clones (C2) accumulated much less or only very negligible amounts of ajmalicine. The possible role of the T(L)-DNA and T(R)-DNA genes on growth and alkaloid accumulation in these root clones is discussed.

Alkaloids↗