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PubMed1992

Expression of cowpea mosaic virus coat protein precursor in transgenic tobacco plants.

Tobacco, Nicotiana tabacum L., supports cowpea mosaic virus (CPMV) replication and cell-to-cell movement, and thus may serve as a model system to study coat protein-mediated protection against CPMV. A chimeric gene consisting of the cauliflower mosaic virus 35S promoter, CPMV 60K coat proteins-precursor (CP-P) coding region, and the nopaline synthase polyadenylation signal was transferred to tobacco cv. Burley 21 via the Agrobacterium tumefaciens binary vector system. Gene integration and expression in the transgenic tobacco plants were confirmed by Southern and RNA dot blot analyses. Accumulation of CPMV 60K CP-P in transgenic plants, up to 2 micrograms/g of wet weight tissue, was detected by ELISA and Western blots. The results of Western blots and immunosorbent electron microscopy further indicated that CPMV CP-P neither undergoes autoproteolysis to generate the mature viral coat proteins nor assembles into virus-like capsids, suggesting that processing of the CP-P may be required for virus assembly. Because CPMV neither induces symptoms in tobacco nor moves systemically, evaluation of the reactions of the transgenic plants to virus inoculation was based on virus accumulation in the inoculated leaves. Results from such infectivity experiments did not differentiate between CP-P expressers and vector-transformed plants. The transgenic tobacco plants expressing CP-P should provide valuable material for investigating comovirus polyprotein processing and capsid assembly in vivo.

Blotting, Southern↗
PubMed1992

The nucleotide sequence of an infectious insect-transmissible clone of the geminivirus Panicum streak virus.

The infectious genome of a Kenyan isolate of Panicum streak virus (PSV) has been cloned and sequenced. Infection of host plants was done using an Agrobacterium binary vector containing a partial repeat of the genome. Progeny virus from resultant infections proved to be transmissible by the leafhopper Cicadulina mbila (Naude). Comparisons of the amino acid sequences of PSV DNA-encoded proteins with those of previously characterized geminiviruses infecting monocotyledonous plants, including maize streak virus, revealed high levels of identity. The evolutionary relationship between PSV and other geminiviruses infecting monocotyledons is discussed.

Amino Acid Sequence↗
PubMed1990

Homologous recombination in plant cells after Agrobacterium-mediated transformation.

A single amino-acid change in the acetolactate synthase (ALS) protein of tobacco confers resistance to the herbicide chlorsulfuron. A deleted, nonfunctional fragment from the acetolactate synthase gene, carrying the mutant site specifying chlorsulfuron resistance plus a closely linked novel restriction site marker, was cloned into a binary vector. Tobacco protoplasts transformed with Agrobacterium tumefaciens carrying this vector yielded chlorsulfuron-resistant colonies. DNA gel blot analysis of DNA from these colonies suggested that in three transformants homologous recombination had occurred between the endogenous ALS gene and the deleted ALS gene present in the incoming T-DNA. Plants were regenerated from these chlorsulfuron-resistant colonies, and in two of the transformants, genetic analysis of their progeny showed that the novel gene segregated as a single Mendelian locus. Possible models for the generation of these recombinant plants are discussed.

Acetolactate Synthase↗
PubMed1986

Activity of T-DNA borders in plant cell transformation by mini-T plasmids.

By using a binary vector system, we examined the requirements for border sequences in T-DNA transformation of plant genomes. Mini-T plasmids consisting of small replicons with different extents of pTiT37 T-DNA were tested for plant tumor-inducing ability in Agrobacterium tumefaciens strain LBA4404 containing helper plasmid pAL4404 (which encodes virulence genes needed for T-DNA transfer). Assays of these bacteria on carrot disks, Kalanchoë leaves, and SR1 Nicotiana tabacum plantlets showed that mini-T plasmid containing full length T-DNA including left and right borders was highly virulent, as were mini-T plasmids containing all onc (oncogenicity) genes and only the right border. In contrast, mini-T plasmids containing all onc genes and only the left border induced tumors only rarely, and a mini-T plasmid containing all onc genes but no T-DNA borders was completely avirulent. Southern hybridization analyses of tumor DNA showed that T-DNA border sequences delimited the extent of the two-border mini-T plasmid transferred and integrated into the plant genome. When only one T-DNA border was present, it formed one end of the transferred DNA, and the other end mapped in the vector sequences. The implications of these results for the mechanism of T-DNA transfer and integration are discussed.

Cell Transformation, Neoplastic↗
PubMed1986

The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA.

We used a binary-vector strategy to study the hypervirulence of Agrobacterium tumefaciens A281, an L,L-succinamopine strain. Strain A281 is hypervirulent on several solanaceous plants. We constructed plasmids (pCS65 and pCS277) carrying either the transferred DNA (T-DNA) or the remainder of the tumor-inducing (Ti) plasmid (pEHA101) from this strain and tested each of these constructs in trans with complementary regions from heterologous Ti plasmids. Hypervirulence on tobacco could be reconstructed in a bipartite strain with the L,L-succinamopine T-DNA and the vir region on separate plasmids. pEHA101 was able to complement octopine T-DNA to hypervirulence on tobacco and tomato plants. Nopaline T-DNA was complemented better on tomato plants by pEHA101 than it was by its own nopaline vir region, but not to hypervirulence. L,L-Succinamopine T-DNA could not be complemented to hypervirulence on tobacco and tomato plants with either heterologous vir region. From these results we suggest that the hypervirulence of strain A281 is due to non-T-DNA sequences on the Ti plasmid.

Amino Acids↗
PubMed1987

Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens A281.

Agrobacterium tumefaciens A281 induces large, rapidly appearing tumors on a variety of plants and has a wider host range than other strains of A. tumefaciens. By using Tn3HoHo1 transposon mutagenesis and complementation analysis, a 2.5-kilobase DNA fragment which is responsible for the supervirulence phenotype was identified in the virulence (vir) region of the Ti plasmid. This fragment contains the virG locus, as well as the 3' end of the virB operon. A clone of this fragment conferred the supervirulence phenotype on A348, a nonsupervirulent strain. The increased virulence was correlated with an increased expression of vir genes, which could be achieved by introducing an extra copy of the transcriptional activator virG or the supervirulence region for maximum virulence. The virulence of the supervirulent strain A281 could be increased even further if the entire virB operon was added in addition to the virG operon. A plasmid, pToK47, containing virB and virG increased the virulence of all A. tumefaciens strains into which the plasmid was introduced. These data suggest that a highly virulent binary vector system can be constructed which might prove especially useful in the transformation of certain higher plants.

Cloning, Molecular↗
PubMed2025

Construction and Segmental Reconstitution of Full-Length Infectious Clones of Milk Vetch Dwarf Virus.

The construction of infectious clones (ICs) is essential for studying viral replication, pathogenesis, and host interactions. Milk vetch dwarf virus (MDV), a nanovirus with a multipartite, single-stranded DNA genome, presents unique challenges for IC development due to its segmented genome organization. To enable functional analysis of its genome, we constructed full-length tandem-dimer-based ICs for all eight MDV genomic segments. Each segment was cloned into a binary vector and co-delivered into Nicotiana benthamiana, Nicotiana tabacum, Vicia faba, and Vigna unguiculata plants via Agrobacterium-mediated inoculation. Systemic infection was successfully reconstituted in all host plants, with PCR-based detection confirming the presence of all viral segments in the infected leaves of nearly all tested plants. Segmental accumulation in infected plants was quantified using qPCR, revealing non-equimolar distribution across hosts. This study establishes the first complete IC system for MDV, enabling reproducible infection, replication analysis, and quantitative segment profiling. It provides a foundational tool for future molecular investigations into MDV replication, host interactions, and viral movement, advancing our understanding of nanovirus biology and transmission dynamics.

Nicotiana↗
PubMed2025

Development of a plant-based vaccine against brucellosis: stable expression of Brucella abortus OMP25 in transgenic tobacco.

Brucellosis, caused by Brucella species, is a global threat to livestock farming, resulting in economic losses and socio-economic challenges, particularly in rural areas. Despite its impact, no licensed human vaccines are available. Animal vaccination remains the most cost-effective control method, but traditional vaccine production is expensive. Edible vaccines, using plants as bioreactors to produce immunogenic antigens, offer a low-cost alternative by eliminating complex purification processes. This study developed a transgenic plant by expressing the Brucella abortus outer membrane protein OMP25 in tobacco plants. OMP25, a conserved transmembrane protein with high immunogenicity, was cloned into a Gateway pDONR vector via a Boundary Pairing reaction and transferred to a binary destination vector via a Left-Right reaction. The destination vector was introduced into Agrobacterium tumefaciens and subsequently used for Agrobacterium-mediated transformation of tobacco plants. Transgenic plants were selected on media containing kanamycin, and the expression of the transgene was verified through the fluorescence of green fluorescent protein. Microcallus formation and shoot development on selective media confirmed kanamycin resistance and the successful integration of the transgene. After phenotypic selection, genomic DNA was extracted from transgenic plants and analyzed by PCR (Polymerase Chain Reaction) using primers specific to the OMP25 gene. Positive PCR results validated the successful integration of the OMP25 gene into the plant genome. Gene expression was further confirmed at the RNA level through real-time quantitative PCR (qRT-PCR) and at the protein level via Western blot analysis. Future studies will evaluate immune responses in animal models. This approach demonstrates the potential for low-cost, effective vaccines to combat brucellosis, addressing critical economic and public health challenges.

Plants, Genetically Modified↗
PubMed1989

Phleomycin resistance as a dominant selectable marker for plant cell transformation.

Tobacco cells are sensitive to bleomycin and phleomycin. The Tn5 and the Streptoalloteichus hindustanus (Sh) bleomycin resistance ('Ble') genes conferring resistance to these antibiotics have each been inserted into two plant expression vectors. They are flanked by the nopaline synthase (nos) or the cauliflower mosaic virus (CaMV) 35S promoters on one side, and by the nos polyadenylation signal on the other. These four chimaeric genes were introduced into the binary transformation vector pGA 492, which were thereafter mobilized into Agrobacterium tumefaciens strain LBA 4404. The resulting strains were used to transform Nicotiana tabacum cv. Xanthi nc using the leaf disc transformation procedure. In all cases, phleomycin- and bleomycin-resistant tobacco plants were regenerated from transformed cells under selective conditions; however, the highest frequency of rooted plants was obtained when transformation was carried out with the 'Sh Ble' gene under the control of the 35S promoter. Phleomycin resistance was stably transmitted to sexual offspring as a dominant nuclear trait as confirmed by Southern blotting.

Bleomycin↗
PubMed2024

Proteome-scale prediction of molecular mechanisms underlying dominant genetic diseases.

Many dominant genetic disorders result from protein-altering mutations, acting primarily through dominant-negative (DN), gain-of-function (GOF), and loss-of-function (LOF) mechanisms. Deciphering the mechanisms by which dominant diseases exert their effects is often experimentally challenging and resource intensive, but is essential for developing appropriate therapeutic approaches. Diseases that arise via a LOF mechanism are more amenable to be treated by conventional gene therapy, whereas DN and GOF mechanisms may require gene editing or targeting by small molecules. Moreover, pathogenic missense mutations that act via DN and GOF mechanisms are more difficult to identify than those that act via LOF using nearly all currently available variant effect predictors. Here, we introduce a tripartite statistical model made up of support vector machine binary classifiers trained to predict whether human protein coding genes are likely to be associated with DN, GOF, or LOF molecular disease mechanisms. We test the utility of the predictions by examining biologically and clinically meaningful properties known to be associated with the mechanisms. Our results strongly support that the models are able to generalise on unseen data and offer insight into the functional attributes of proteins associated with different mechanisms. We hope that our predictions will serve as a springboard for researchers studying novel variants and those of uncertain clinical significance, guiding variant interpretation strategies and experimental characterisation. Predictions for the human UniProt reference proteome are available at https://osf.io/z4dcp/.

Humans↗
PubMed1990

A Markovian model for comparing incidences of side effects.

For clinical trials that entail observations at successive visits for the occurrence of a side effect, this paper considers a likelihood-based method to compare side effect incidence rates. The method, which employs the assumption of a Markov chain of order one for the vectors of binary responses, handles missing data due to premature withdrawals. An actual numerical example and a simulated example illustrate the technique.

Clinical Trials as Topic↗
PubMed1990

Agrobacterium-mediated DNA transfer in sugar pine.

DNA transfer using Agrobacterium tumefaciens has been demonstrated in sugar pine, Pinus lambertiana Dougl. Shoots derived from cytokinin-treated cotyledons formed galls after inoculation with A. tumefaciens strains containing the plasmid pTiBo542. A selectable marker, neomycin phosphotransferase II, conferring resistance to kanamycin, was transferred into sugar pine using a binary armed vector system. Callus proliferated from the galls grew without hormones and in some cases, kanamycin-resistant callus could be cultured. Southern blots provided evidence of physical transfer of T-DNA and the nptII gene. Expression of the nptII gene under control of the nos promoter was demonstrated by neomycin phosphotransferase assays. Several aspects of DNA transfer were similar to those previously observed in angiosperms transformed by A. tumefaciens. This is the first evidence for DNA transfer by Agrobacterium in this species and the first physical evidence for transfer in any pine. These results bring us closer to genetic engineering in this commercially important genus of forest trees.

DNA↗
PubMed1990

Aminoglycoside-3''-adenyltransferase confers resistance to spectinomycin and streptomycin in Nicotiana tabacum.

The bacterial gene aadA encodes the enzyme aminoglycoside-3"-adenyltransferase that confers resistance to spectinomycin and streptomycin in Escherichia coli. Chimeric genes have been constructed for expression in plants, and were introduced into Nicotiana tabacum by Agrobacterium binary transformation vectors. Spectinomycin or streptomycin in selective concentrations prevent greening of N. tabacum calli. Transgenic clones, however, formed green calli on selective media containing spectinomycin, streptomycin, or both drugs. Resistance was inherited as a dominant Mendelian trait in the seed progeny. Resistance conferred by the chimeric aadA gene can be used as a color marker similar to the resistance conferred by the streptomycin phosphotransferase gene to streptomycin.

Base Sequence↗
PubMed1987

High meiotic stability of a foreign gene introduced into tobacco by Agrobacterium-mediated transformation.

Two lines of transgenic Nicotiana tabacum transformed to kanamycin resistance by means of a binary Agrobacterium vector containing a nos-npt gene were investigated over three generations. Southern hybridization and crossing analyses revealed that a single copy of T-DNA had integrated in each line and that the kanamycin resistance was regularly transmitted to the progeny as a monogenic dominant trait. Homozygous transgenic plants were fully fertile, morphologically normal and did not significantly differ from wild-type plants in the quantitative characters examined (plant height, flowering time). The two lines showed very low, but significantly different levels of meiotic instability: kanamycin-sensitive plants occurred among backcross progeny from homozygous transgenic plants with frequencies of 6/45,000 and 25/45,000, respectively. The sensitive plants arose independently of each other and thus resulted from meiotic rather than mitotic events. These findings demonstrate for the first time that integrated foreign genes can be transmitted to progeny with the high degree of meiotic stability required for commercial varieties of crop plants. They emphasize the importance of non-homologous integration and of avoiding co-integration of inactive gene copies for achieving meiotically stable transformants.

Genetic Vectors↗
PubMed1991

The nucleotide sequence and genome structure of the geminivirus miscanthus streak virus.

A tandem dimer of miscanthus streak virus (MiSV) DNA was inserted into the T-DNA of the binary plasmid vector pBIN19 and agroinoculated into several monocotyledonous plants (monocots) using Agrobacterium tumefaciens or A. rhizogenes. Disease symptoms and geminate particles were produced in maize and Panicum milaceum plants, and MiSV-specific double-stranded and single-stranded DNAs were found in these plants. The nucleotide sequence of the infectious MiSV clone, consisting of 2672 nucleotides, was determined. Four open reading frames (ORFs) for proteins of Mr greater than 10K were identified, two (V0 and V2) in the virus (+) sense and two (C1 and C2) in the complementary (-) sense, although C2 did not have an ATG start codon. Unlike other geminiviruses infecting monocots, complementary-sense ORFs did not overlap. Potential splicing donor and acceptor sites were identified in the sequence of the border region between the C terminus of ORF C1 and the N terminus of ORF C2. Amino acid sequences predicted from three (V2, C1 and C2) of these ORFs showed significant homology with the corresponding ORFs of other geminiviruses infecting monocots. A fifth ORF (V1), which showed some homology with ORF V1 of other monocot-infecting geminiviruses despite having a coding capacity for a product of Mr 8.8K, was found just upstream of ORF V2 as observed in those geminiviruses. ORF V0 showed no significant homology with ORFs present in any other geminiviruses. A mutation of V0 indicated that the C-terminal 30% of this ORF was not necessary for infection in maize, but that sequences around the mutated LspI site might have some regulatory role.

Amino Acid Sequence↗
PubMed1991

Insect resistance of transgenic tobacco plants expressing delta-endotoxin gene of Bacillus thuringiensis.

The initiative B.thuringiensis delta-endotoxin (Bt toxin) gene clones TH12 and TH48 contain two different classes of homologous genes, the 5.3 kb class and 6.6 kb class, respectively. Bt toxin genes of both classes, modified at the 5'-end and truncated at the 3'-end, can still be expressed to produce the insecticidal, truncated toxin proteins in E. coli. The modified Bt toxin genes were inserted into the plant binary expression vector pBin 437 (a derivative plasmid of pBin 19) and were transferred into tobacco by Ti plasmid-mediated gene transfer system. Southern blot and DNA slot blot analysis indicate that the Bt toxin genes have been integrated into tobacco genome at a copy number of 1 to 5. Northern blot analysis of polyA+ RNAs from progeny of the transgenic plants revealed that Bt toxin genes of both 5.3 kb and 6.6 kb classes were expressed in transgenic plants, though the transcripts were degraded to RNAs of lower molecular weights. In insecticidal test, 5 plants from the progeny of 5.3 kb class gene-transformed SR1 tobacco plants and 3 plants from those of 6.6 kb class gene-transformed plants were found to be toxic to the testing larvea of H.assulta. In comparison with the control, mortality of the insects fed on transgenic plants reached 40-50% and the growth of the survived insects was remarkably inhibited. These results indicate that the modified Bt genes of the 5.3 kb and 6.6 kb classes were expressed in transgenic plants and could confer on the transgenic plants a new character of insect resistance.

Animals↗
PubMed1990

Binary conjugational transfer system of vectors pRK290 and pLAFRI is proficient both ways between Escherichia coli and Rhizobium.

Wide host range vector plasmids pRK290 and pLAFRI carrying genomic fragments of Rhizobium are transferable both ways between R. meliloti and R. leguminosarum cells on the one hand and to E. coli cells on the other, in triparental matings involving E. coli cells carrying pRK2013, the helper for Tra functions to the vector plasmids. The vector plasmids pRK290 and pLAFRI can be employed for recovering clones harbored by R. leguminosarum and R. meliloti by transfer to Rhizobium cells by direct matings of the library with them.

Cloning, Molecular↗
PubMed1992

Development and characterization of a generalized gene tagging system for higher plants using an engineered maize transposon Ac.

This report describes a series of transposon tagging vectors for dicotyledonous plants based on the maize transposable element Ac. This binary system includes the transposase (Ts) and the tagging element (Ds) on separate T-DNA vectors. Ts elements include versions in which transcription is driven either by the endogenous Ac promoter or by the cauliflower mosaic virus (CaMV) 35S promoter. Ds tagging element includes a gene conferring methotrexate (Mtx) resistance for selection and a supF gene to facilitate cloning of tagged sequences. The Ds element is flanked by a CaMV 35S promoter and the beta-glucuronidase (GUS) coding sequence so that GUS expression occurs upon excision of the element. We have transformed these Ts and Ds elements into tobacco and demonstrated that the Ts is functional with either promoter, and that the artificial Ds elements are capable of transposition. The amount of excision was found to depend upon both the individual Ts and Ds primary transformants used. Somatic excision of Ds was seen in up to 100% of progeny seedlings containing Ts and Ds. Germinal excision was detected in up to 48% of the progeny of plants containing both elements. Hence, this system can generate a sufficient number of events to be useful in gene tagging.

Base Sequence↗