Binary Vector System Research Articles

Bax-Induction Gene Therapy of Pancreatic Cancer

Background. Bax is a strong pro-apoptotic gene that induces programmed cell death when expressed. Human telomerase reverse transcriptase (hTERT) is the catalytic subunit for telomerase, an enzyme found to be active in more than 85% of human cancers. Recently, a binary adenoviral system (Ad/GT- Bax + Ad/hTERT-GV16) was constructed using the hTERT promoter to induce Bax gene expression in tumor cells. Methods. To test whether human pancreatic tumor cells would respond to this system of Bax-induced apoptosis, we compared the effects of Bax gene induction with that of LacZ gene induction using the same binary system. Results. Lysates of the human pancreatic cell lines PANC-28, MIA PaCa-2, and BxPC-3 showed significantly elevated levels of human telomerase using the PCR-based TRAP assay. As early as 24 h after treatment with Bax-induction gene therapy, growth inhibition was observed. Overexpression of the Bax protein was confirmed by Western blotting. Extensive apoptosis on FACS analysis at 48 h was seen after Bax induction. In addition, cytosolic cytochrome c levels increased compared to mitochondrial levels after Bax induction. Levels of caspase-3, a key downstream enzyme involved in apoptosis, also increased significantly compared to controls after treatment. None of these effects were seen with LacZ. Conclusion. Our results suggest that the binary adenoviral vector system, Ad/GT- Bax + Ad/hTERT-GV16, induces high levels of Bax expression that induce apoptosis in human pancreatic cancer cells.

Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system.

We have achieved routine transformation of maize (Zea mays) using an Agrobacterium tumefaciens standard binary (non-super binary) vector system. Immature zygotic embryos of the hybrid line Hi II were infected with A. tumefaciens strain EHA101 harboring a standard binary vector and cocultivated in the presence of 400 mg L-1 L-cysteine. Inclusion of L-cysteine in cocultivation medium lead to an improvement in transient beta-glucuronidase expression observed in targeted cells and a significant increase in stable transformation efficiency, but was associated with a decrease in embryo response after cocultivation. The average stable transformation efficiency (no. of bialaphos-resistant events recovered per 100 embryos infected) of the present protocol was 5.5%. Southern-blot and progeny analyses confirmed the integration, expression, and inheritance of the bar and gus transgenes in R0, R1, and R2 generations of transgenic events. To our knowledge, this represents the first report in which fertile, stable transgenic maize has been routinely produced using an A. tumefaciens standard binary vector system.

Agrobacterium‐mediated T‐DNA transfer and integration into the chromosome of Streptomyces lividans

Summary Agrobacterium tumefaciens is the prototype of a prokaryotic organism transmitting DNA across natural kingdom barriers into higher cells. In nature, a specific segment (T-DNA) of the resident Ti plasmid is transferred from this bacterium into plant cells and integrated into the plant cell genome. Expression of the integrated oncogenes contained in the T-DNA results in the tumour disease known as crown gall. Besides plants, the range of transformable recipients is broad and includes fungi and mammalian cells. We now show further extension of this host range, whereby the actinomycete Streptomyces lividans is also a recipient of the T-DNA. A. tumefaciens cells containing a binary vector system with a vir helper plasmid, pUCD2614, and a T-DNA donor plasmid, pUCD5801, were co-cultured with S. lividans hyphae. A. tumefaciens-S. lividans aggregate when the vir genes are induced with acetosyringone, resulting in the transfer of the T-DNA, as evidenced by the formation of transconjugants containing T-DNA genetic markers and the appearance of the T-DNA in these transconjugants. Close examination of the interacted cells revealed a presumably coiled thread-like interconnection with an average width of approximately 30 nm between A. tumefaciens and S. lividans. This interconnecting structure is dependent on virB genes and appears only under the same conditions as that required for T-pilus formation. Insertion of the T-DNA via A. tumefaciens into the S. lividans genome provides a useful genetic tool for generating novel mutants.

Alkaloid production in Duboisia hybrid hairy root cultures overexpressing the pmt gene

Putrescine:SAM N-methyltransferase (PMT) catalyses the N-methylation of the diamine putrescine to form N-methylputrescine, the first specific precursor of both tropane and pyridine-type alkaloids, which are present together in the roots of Duboisia plants. The pmt gene of Nicotiana tabacum was placed under the regulation of the CaMV 35S promoter and introduced into the genome of a scopolamine-rich Duboisia hybrid by a binary vector system using the disarmed Agrobacterium tumefaciens strain C58C1 carrying the rooting plasmid pRiA4. The presence of the foreign gene in kanamycin-resistant hairy roots and its overexpression were confirmed by polymerase chain reaction and Northern blot analysis respectively. The N-methylputrescine levels of the resulting engineered hairy roots increased (2–4-fold) compared to wild type roots, but there was no significant increase in either tropane or pyridine-type alkaloids.

Generation of selectable marker-free transgenic rice using double right-border (DRB) binary vectors

Currently employed transformation systems require selectable marker genes encoding antibiotic or herbicide resistance, along with the gene of interest (GOI), to select transformed cells from among a large population of untransformed cells. The continued presence of these selectable markers, especially in food crops such as rice (Oryza sativa L.), is of increasing public concern. Techniques based on DNA recombination and Agrobacterium-mediated co-transformation with two binary vectors in a single or two different Agrobacterium strains, or with super-binary vectors carrying two sets of T-DNA border sequences (twin T-DNA vectors), have been employed by researchers to produce selectable marker-free (SMF) transgenic progeny. We have developed a double right-border (DRB) binary vector carrying two copies of T-DNA right-border (RB) sequences flanking a selectable marker gene, followed by a GOI and one copy of the left border sequence. Two types of T-DNA inserts, one initiated from the first RB containing both the selectable gene and the GOI, and the other from the second RB containing only the GOI, were expected to be produced and integrated into the genome. In the subsequent generation, these inserts could segregate away from each other, allowing the selection of the progeny with only the GOI. We tested this vector using two selectable marker genes and successfully obtained progeny plants in which the second selectable marker gene segregated away from the first. Using the DRB binary vector system, we recovered SMF transgenic lines containing a rice ragged stunt virus (RRSV)-derived synthetic resistance gene in the rice cultivars Jarrah and Xiu Shui. Approximately 36–64% of the primary transformants of these cultivars yielded SMF progeny. Among SMF Jarrah transgenic progeny <50% of plants contained the RRSV transgene. Thus, we have developed an efficient vector for producing SMF plants that allows straightforward cloning of any GOIs in comparison with the published ‘twin T-DNA’ vectors.

The Arabidopsis phytochrome B gene influences growth of the apple rootstock M26

The apple rootstock M26 (Malus domestica) was infected with a binary vector system of Agrobacterium tumefaciens carrying the neomycin phosphotransferase II and Arabidopsis phyB genes. Thirteen transformed clones were obtained from 329 infected leaves. Five of the clones had a single copy integration, six clones had two copies, one clone had five copies and one of the clones had eight copies of the phyB gene integrated. No differences in rooting were found between transformed and untransformed plants. The stem length was reduced in nine of the 13 transgenic clones, and shoot, root and plant dry weights were reduced in all transformed clones compared with untransformed control plants. Northern analysis showed that the Arabidopsis phyB gene was expressed in the transformed clones.

The basal-level expression of the cytochrome P450(BM-1) gene is negatively affected by the bm1P1 gene of Bacillus megaterium.

Evidence against the Bm1P1 protein as a positive transcription factor for barbiturate-mediated induction of cytochrome P450(BM-1) in Bacillus megaterium has been provided in our previous report. In the present study, we show that the basal-level expression of the P450(BM-1) promoter-xylE transcriptional fusion is significantly reduced when the bm1P1 gene is present on the same plasmid. Moreover, disruption of the chromosomal bm1P1 gene results in enhanced expression of the P450(BM-1) promoter-xylE fusion on a multicopy plasmid. By using a binary vector system, we found that expression of the P450(BM-1) promoter-xylE fusion from a high-copy plasmid is substantially repressed by expression of bm1P1 from a low-copy plasmid. Taken together, these results suggest that the basal-level expression of P450(BM-1) is negatively affected by bm1P1. Possible mechanisms for this control are discussed.

A binary adenoviral vector system for expressing high levels of the proapoptotic gene bax.

The bax gene plays a critical role in signaling apoptosis and expression through gene transfer may be valuable in the treatment of a variety of apoptosis-related diseases such as cancer. However, constructing an adenoviral vector expressing a bax gene driven by a constitutive promoter has been difficult, presumably because of the gene's high proapoptotic activity. Here we report a system that induces the expression of the bax gene safely by adenovirus-mediated gene cotransfer. Briefly, the system involves an adenoviral vector containing a human bax cDNA driven by a synthetic promoter consisting of five GAL4-binding sites and a TATA box (GT). This vector expresses a minimal background level of bax protein in cultured mammalian cells thus preventing apoptosis of packaging cells, however, expression of the bax gene can be induced substantially in vitro and in vivo by transferring it into target cells along with an adenoviral vector expressing the transactivator, fusion protein GAL4/VP16. Extensive apoptosis was observed after induction of the bax gene both in cultured human lung carcinoma cells and in the livers of Balb/c mice. Our results suggest that this GAL4 gene regulatory system provides an alternative approach to constructing viral vectors that express potentially toxic genes. Gene Therapy (2000) 7, 75-79.

Activity of the yeast mevalonate kinase promoter in transgenic tobacco

The coding region of the yeast mevalonate kinase gene ( ERG12) under the control of the cloned 564 bp promoter was introduced in tobacco ( Nicotiana tabacum cv. Paraguay Bell) using an Agrobacterium tumefaciens binary vector system. In this study, we provide evidence that the ERG12 promoter is sufficient to direct MK expression in agrobacteria and in plant cells. The tissue-specific pattern of expression directed by the ERG12 promoter was investigated by analysis of β-glucuronidase (GUS) activity in tobacco. A preferential expression of a ERG12-GUS fusion gene was observed in fast dividing cells of calli during regeneration and in meristematic tissues such as root-tips, lateral root primordia and the shoot apexes of growing plantlets. Histochemical tests and measurements of MK activity emphasize a temporal regulation of MK expression, leading to phenotypical modifications: acceleration of regenerating processes, peculiar flowering and starch accumulation in roots. Taken together, these results provide additional clues as to the possible functional role of MVA derivatives (i.e. some cytokinins or prenylated proteins) in cell cycle control.

Graded transcriptional response to different concentrations of a single transactivator.

Threshold mechanisms of transcriptional activation are thought to be critical for translating continuous gradients of extracellular signals into discrete all-or-none cellular responses, such as mitogenesis and differentiation. Indeed, unequivocal evidence for a graded transcriptional response in which the concentration of inducer directly correlates with the level of gene expression in individual eukaryotic cells is lacking. By using a novel binary tetracycline regulatable retroviral vector system, we observed a graded rather than a threshold mechanism of transcriptional activation in two different model systems. When polyclonal populations of cells were analyzed at the single cell level, a dose-dependent, stepwise increase in expression of the reporter gene, green fluorescent protein (GFP), was observed by fluorescence-activated cell sorting. These data provide evidence that, in addition to the generally observed all-or-none switch, the basal transcription machinery also can respond proportionally to changes in concentration of extracellular inducers and trancriptional activators.

Expression of the yeast mevalonate kinase gene in trangenic tobacco

The coding region of the yeast mevalonate kinase gene (ERG12), under the control of the cauliflower mosaic virus (CaMV) 35S promoter, has been inserted in tobacco (Nicotiana tabacum cv. Paraguay Bell) using an Agrobacterium tumefaciens binary vector system. Integration and expression of the ERG12 chimaeric gene was demonstrated in several independent transformants in which specific mevalonate kinase (MK) activity in young plantlets was increased by about 60% on average. The expression of this MK gene was accompanied by phenotypical modifications, such as acceleration of regenerating processes, lateral bud growth, and peculiar flowering behaviour. A higher chlorophyll content all along the plant development, paralleled by an unusual starch accumulation in the leaves of young plantlets and, later, in roots of full-grown plants, was also detected. Overexpression of the MK gene led also to a stronger inhibition of cytokinin-induced plant growth by methyl jasmonate in transgenic plants. All these events may be interpreted as a possible modification of the hormonal balance in transgenic tobaccos.

Development of a binary vector system for plant transformation based on the supervirulent Agrobacterium tumefaciens strain Chry5.

This report describes the disarming of Agrobacterium tumefaciens Chry5, a strain highly tumorigenic on soybean. Disarming was achieved by removing an approximately 16.5-kb segment of the 285-kb Ti plasmid pTiChry5, including approximately 4 kb of the oncogenic T-DNA and an extended region right of the T-DNA, and replacing it with a gene for carbenicillin resistance, through homologous recombination. The deletion was confirmed with Southern analysis, and the loss of tumorigenicity was verified in tobacco and tomato plant stem inoculation assays. The deletion mutant, named KYRT1, successfully transferred the β-glucuronidase (GUS) gene into tobacco leaf tissue, producing GUS-expressing callus which could be regenerated into viable plants. In a comparative study, the transformation efficiency of A. tumefaciens KYRT1, GV3850, and EHA105 was assayed by inoculating cotyledonary node explants. The results of this study revealed that, in a binary vector system, KYRT1 is equally or more effective than EHA105 or GV3850 at delivering DNA into soybean.

Transgenic melon (cucumis melo L.) and potential for expression of novel proteins important to food industry

Melon (Cucumis melo L.) is an important fruit crop cultivated widely in every region of the world. Our laboratory is targeting this species for production of novel proteins important to food industry. Prior to expression of protein of interest in transgenic melon an efficient genetic transformation system has to be developed. In this context we are testing a wide variety of promoters fused to reporter gene for β‐glucuronidase (GUS) for expression specifically in melon fruits. In this study in melon, salicylic acid‐inducible promoter region of pathogenesis‐related protein gene (PR1a) of tobacco fused to β‐glucuronidase (GUS) gene was introduced into melon via Agrobacterium‐mediated gene transfer using a binary vector system. Gene transfer was effective when Agrobacterium virulence factors like acetosyringone (100 μM) and low pH (5.2) were provided during the co‐culture step. Transformed shoots were recovered from benzyladenine‐induced cut cotyledons using kanamycin gene as a selective marker. Regeneration of shoots from cotyledons was stimulated by providing 10 mM proline in the shoot organogenesis medium. Southern and Northern blot analysis of transformants confirmed the presence of β‐glucuronidase gene in two selected clones J‐3 and PR‐G. The transformants also showed high β‐glucuronidase activity after salicylic acid treatment. Thiamine, a previously known inducer of pathogenesis‐related protein, stimulated β‐glucuronidase in J‐3 but not PR‐G melon transformants tested in this study. These studies showed that tobacco PR1a promoter region can be expressed in melon and it was stimulated by salicylic acid. This indicates the potential to use the promoter region of tobacco PR1a for genetic improvement of melon for specific food processing‐related characteristics or for expression of novel food‐related proteins. The promoter region could be used to drive specific target genes under stress or salicylic acid induced conditions.

Agrobacterium-Mediated Transformation of Lettuce (Lactucae sativa)

Lettuce cultivars. Diana and Cobham Green were used for transformation using the binary vector system of Agrobacterİum tumefaciens. A \beta-1, 3-glucanase gene from Arthrobacter driven by the 35 S prometer and NPT 11 gene expressed from Nos promoter were involved as scorible and selectable genes. respectively. Transformations were obtained at a high frequency particulary with cultivar Cobham Green (80%). Transformation was confirmed by using Southern and Northern blot analysis. NPT 11 enzyme assay and segregation of resistance to kanamycin. Our results showed that this protocol can be efficiently used for transforming Iettuce.

Proteinase inhibitor II gene in transgenic poplar: Chemical and biological assays

Transgenic poplar lines were developed to investigate the role of a proteinase inhibitor in pest resistance of woody plants. Using an Agrobacterium binary vector system, the clone ‘Hansen’ ( Populus alba L. × P. grandidentata Michx.) was transformed with chimeric genes containing the coding region of potato proteinase inhibitor II ( PIN2) linked to either a bacterial nopaline synthase ( nos) or a cauliflower mosaic virus (35 S) promoter. All transferred DNA also contained a selectable marker in the form of a nos promoter linked to a neomycin phosphotransferase II ( NPT II) structural gene. The presence of the transferred PIN2 and NPT II sequences in poplar was confirmed for nine transgenic lines using polymerase chain reaction (PCR). Expression of PIN2 in leaves of transgenic poplar was demonstrated by enzyme-linked immunosorbent assays (ELISAs) and western blots. Two unique polypeptides from transgenic poplar, of ca 8 kDa and ca 12 kDa, indicate that PIN2 was translated appropriately. Resistance to the imported willow leaf beetle was tested in laboratory bioassays. The untransformed clone ‘Hansen’ and 11 transgenic lines were submitted to freshly hatched larvae to determine effects on pupal weight, larval development time and leaf area consumed. A significant difference from the untransformed clone in leaf area consumed was detected in one transgenic line, Tr665. Trends were indicated for several other transgenic lines for the other parameters.

Transformation of Perilla frutescens var. crispa Using an Agrobacterium-Ri Binary Vector System.

Transgenic hairy roots of Perilla frutescens var. crispa were obtained using an Agrobacterium-Ri binary vector system. The chimeric uidA gene encoding β-glucuronidase (GUS) flanked by the TR2′ promoter for mannopine synthase was introduced in the plant genome by Agrobacterium-mediated transformation by means of scratching stems of young plants. The adventitious roots which arose on the infected sites were excised, and the several clones of root tissue could be maintained on hormone-free agar medium. The transgenic states of these clones were confirmed by Southern blot hybridization. Expression of the uidA gene in the transgenic tissues was indicated by in vitro enzyme assay and histochemical staining of GUS activity. More than half of hairy roots which emerged were confirmed to be doubly transformed with T-DNAs from Ri and binary Ti plasmids.

Transgenic fertile Scoparia dulcis L., a folk medicinal plant, conferred with a herbicide-resistant trait using an Ri binary vector

Transgenic herbicide-resistant Scoparia dulcis plants were obtained by using an Ri binary vector system. The chimeric bar gene encoding phosphinothricin acetyltransferase flanked by the promoter for cauliflower mosaic virus 35S RNA and the terminal sequence for nopaline synthase was introduced in the plant genome by Agrobacterium-mediated transformation by means of scratching young plants. Hairy roots resistant to bialaphos were selected and plantlets (R0) were regenerated. Progenies (S1) were obtained by self-fertilization. The transgenic state was confirmed by DNA-blot hybridization and assaying of neomycin phosphotransferase II. Expression of the bar gene in the transgenic R0 and S1 progenies was indicated by the activity of phosphinothricin acetyltransferase. Transgenic plants accumulated scopadulcic acid B, a specific secondary metabolite of S. dulcis, in amounts of 15-60% compared with that in normal plants. The transgenic plants and progenies showed resistant trait towards bialaphos and phosphinothricin. These results suggest that an Ri binary system is one of the useful tools for the transformation of medicinal plants for which a regeneration protocol has not been established.

Isolation and characterization of Bacillus subtilis groE regulatory mutants: evidence for orf39 in the dnaK operon as a repressor gene in regulating the expression of both groE and dnaK

An inverted repeat sequence known as CIRCE (controlling inverted repeat of chaperone expression) in the Bacillus subtilis groE operon has been suggested to function as an operator. To identify the regulatory gene directly or indirectly involved in CIRCE-mediated heat-inducible groE expression, B. subtilis WBG2, carrying an integrated groE-bgaB transcription fusion in the amyE locus, was mutagenized. Dark blue colonies formed at 37 degrees C represent mutants which constitutively produce BgaB (a thermostable beta-galactosidase) at high levels. Seven mutants (WBG101 to WBG107) were selected for further characterization. They all overproduced BgaB, GroEL, and DnaK simultaneously at 37 degrees C. These mutants could be restored to normal by introducing a plasmid carrying a functional copy of orf39, the first gene in the B. subtilis dnaK operon. Genomic sequencing of these mutants demonstrated that they all carried a single mutation in orf39. These mutations can be divided into three groups: (i) Gly-307 to Asp, (ii) Ser-122 to Phe, and (iii) Gly-63 to Glu. By using a binary vector system in E. coli, production of ORF39 was found to negatively regulate the expression of groE-bgaB in a CIRCE-specific manner. Under the heat shock condition, the negative regulation mediated by ORF39 was abolished. Mobility shift of the CIRCE-containing probe was also observed with the crude extract prepared from the E. coli strain that overproduced ORF39. Therefore, ORF39 is the negative regulatory factor which regulates both groE and dnaK expression in B. subtilis. It is likely to function as a CIRCE-specific repressor.

Development of insect resistance in tomato plants expressing the δ-endotoxin gene ofBacillus thuringiensis subsp.tenebrionis

A crystal δ-endotoxin gene ofBacillus thuringiensis subsp.tenebrionis (B.t.t.) encoding a coleopteran insect-specific toxin was used to construct a chimeric gene which expressed the toxin in plant cells. Via anAgrobacterium tumefaciens binary vector system, the toxin gene was transferred into tomato cells. From leaf disks recombinant plants were regenerated. Hybridization experiments demonstrated that these plants synthesized toxin-specific mRNA of the expected size. Transgenic tomato plants with the chimericB.t.t. toxin gene contained a 74 kDa protein which cross-reacted with toxin antibodies. The expression caused a significant insecticidal activity of the transgenic tomato plants against Colorado potato beetle larvae.

Developmental specific expression and organelle targeting of the Escherichia coli fabD gene, encoding malonyl coenzyme A-acyl carrier protein transacylase in transgenic rape and tobacco seeds.

In both plants and bacteria, de novo fatty acid biosynthesis is catalysed by a type II fatty acid synthetase (FAS) system which consists of a group of eight discrete enzyme components. The introduction of heterologous, i.e. bacterial, FAS genes in plants could provide an alternative way of modifying the plant lipid composition. In this study the Escherichia coli fabD gene, encoding malonyl CoA-ACP transacylase (MCAT), was used as a model gene to investigate the effects of over-producing a bacterial FAS component in the seeds of transgenic plants. Chimeric genes were designed, so as not to interfere with the household activities of fatty acid biosynthesis in the earlier stages of seed development, and introduced into tobacco and rapeseed using the Agrobacterium tumefaciens binary vector system. A napin promoter was used to express the E. coli MCAT in a seed-specific and developmentally specific manner. The rapeseed enoyl-ACP reductase transit peptide was used successfully, as confirmed by immunogold labelling studies, for plastid targeting of the bacterial protein. The activity of the bacterial enzyme reached its maximum (up to 55 times the maximum endogenous MCAT activity) at the end of seed development, and remained stable in mature transgenic seeds. Significant changes in fatty acid profiles of storage lipids and total seed lipid content of the transgenic plants were not found. These results are in support of the notion that MCAT does not catalyse a rate-limiting step in plant fatty acid biosynthesis.