Electroporation of megaplasmids into Agrobacterium

Mozo, Teresa; Hooykaas, Paul J. J.

doi:10.1007/bf00015085

Cited by 62 publications

(37 citation statements)

References 6 publications

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“…Plasmid LAL21 was isolated from Escherichia coli strain DH5␣ and transformed into A. rhizogenes LBA9402 by electroporation (20). A positive clone, after confirmation by PCR and enzymatic digestion analysis for the presence of the h6h gene, was used for plant transformation.…”

Section: Methodsmentioning

confidence: 99%

“…The pXI was isolated from E. coli strain DH5␣ and transformed into disarmed A. tumefaciens strain C58C1 containing A. rhizogenes Ri plasmid pRiA4 (20). A positive clone, after confirmation by PCR and enzymatic digestion analysis for the presence of both pmt and h6h genes, was used to transform plant tissues for simultaneous expression of pmt and h6h.…”

Section: Methodsmentioning

confidence: 99%

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Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures

Zhang

Ding

Chai

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

243

126

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Scopolamine is a pharmaceutically important tropane alkaloid extensively used as an anticholinergic agent. Here, we report the simultaneous introduction and overexpression of genes encoding the rate-limiting upstream enzyme putrescine N-methyltransferase (PMT) and the downstream enzyme hyoscyamine 6 ␤-hydroxylase (H6H) of scopolamine biosynthesis in transgenic henbane (Hyoscyamus niger) hairy root cultures. Transgenic hairy root lines expressing both pmt and h6h produced significantly higher (P < 0.05) levels of scopolamine compared with the wild-type and transgenic lines harboring a single gene (pmt or h6h). The best line (T 3) produced 411 mg͞liter scopolamine, which was over nine times more than that in the wild type (43 mg͞liter) and more than twice the amount in the highest scopolamine-producing h6h single-gene transgenic line H 11 (184 mg͞liter). To our knowledge, this is the highest scopolamine content achieved through genetic engineering of a plant. We conclude that transgenic plants harboring both pmt and h6h possessed an increased flux in the tropane alkaloid biosynthetic pathway that enhanced scopolamine yield, which was more efficient than plants harboring only one of the two genes. It seems that the pulling force of the downstream enzyme (the faucet enzyme) H6H plays a more important role in stimulating scopolamine accumulation in H. niger whereas the functioning of the upstream enzyme PMT is increased proportionally. This study provides an effective approach for large-scale commercial production of scopolamine by using hairy root culture systems as bioreactors.Agrobacterium ͉ hyoscyamine 6␤-hydroxylase ͉ putrescine N-methyltransferase ͉ scopolamine ͉ transformation

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures

Zhang

Ding

Chai

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

243

126

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“…The amplified fragment was first cloned into the pCRII vector, using a TA Cloning Kit (Invitrogen, Carlsbad CA), and then transferred into plasmid pBIN19. The resultant recombinant (pAtXPD) was introduced into Agrobacterium strain GV3101 by electroporation, as described by Mozo and Hooykaas (1991). Transformed bacteria were selected on Luria-Bertani medium (Sambrook et al, 1989) containing 30 mg L Ϫ1 gentamycin and 60 mg L Ϫ1 kanamycin.…”

Section: Mutant Complementationmentioning

confidence: 99%

Arabidopsis UVH6, a Homolog of Human XPD and Yeast RAD3 DNA Repair Genes, Functions in DNA Repair and Is Essential for Plant Growth

et al. 2003

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To evaluate the genetic control of stress responses in Arabidopsis, we have analyzed a mutant (uvh6-1) that exhibits increased sensitivity to UV light, a yellow-green leaf coloration, and mild growth defects. We have mapped the uvh6-1 locus to chromosome I and have identified a candidate gene, AtXPD, within the corresponding region. This gene shows sequence similarity to the human (Homo sapiens) XPD and yeast (Saccharomyces cerevisiae) RAD3 genes required for nucleotide excision repair. We propose that UVH6 is equivalent to AtXPD because uvh6-1 mutants carry a mutation in a conserved residue of AtXPD and because transformation of uvh6-1 mutants with wild-type AtXPD DNA suppresses both UV sensitivity and other defective phenotypes. Furthermore, the UVH6/AtXPD protein appears to play a role in repair of UV photoproducts because the uvh6-1 mutant exhibits a moderate defect in the excision of UV photoproducts. This defect is also suppressed by transformation with UVH6/AtXPD DNA. We have further identified a T-DNA insertion in the UVH6/AtXPD gene (uvh6-2). Plants carrying homozygous insertions were not detected in analyses of progeny from plants heterozygous for the insertion. Thus, homozygous insertions appear to be lethal. We conclude that the UVH6/AtXPD gene is required for UV resistance and is an essential gene in Arabidopsis.DNA damage is a challenge for all organisms exposed to UV irradiation. UV photoproducts consist primarily of cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidinone dimers (Mitchell and Nairn, 1989;Pfeifer, 1997). These lesions inhibit DNA replication and transcription and also promote mutagenesis (McGregor, 1999). The effects of UV irradiation are especially detrimental in plants, where sunlight is both a source of damage and a requirement for photosynthesis.Increasing evidence suggests that plants repair UVdamaged chromosomes using mechanisms similar to those found in humans (Homo sapiens) and yeast (Saccharomyces cerevisiae). These mechanisms include the nucleotide excision repair (NER) pathway, a process which involves recognition of UV lesions, incision of the damaged strand on both sides of the lesion, removal of the damaged fragment, and repair by gap filling and ligation (Batty and Wood, 2000;de Boer and Hoeijmakers, 2000;Prakash and Prakash, 2000). Several potential plant homologs of human and yeast NER genes have been identified. Genetic analyses of these plant genes, including studies of the phenotypes of plants carrying mutations within these genes, provide support for the idea that the NER pathway is conserved in plants.Lesion recognition during NER involves the homologous heterodimers XPC:HR23B (human) and RAD4:RAD23 (yeast; Balajee and Bohr, 2000;Batty and Wood, 2000;de Boer and Hoeijmakers, 2000;Prakash and Prakash, 2000). The Arabidopsis genome contains potential homologs of both XPC/RAD4 and HR23B/RAD23 (Arabidopsis Genome Initiative, 2000). HR23B expression occurs in Arabidopsis, rice (Oryza sativa), and carrot (Daucus carota), and the carrot gene complements the r...

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“…Dimers of the respective molecules, excised by BamHI, were inserted into the BamHI site of plasmid pBin19. Subsequently, the pBin19 derivatives were transferred by electroporation (30) into Agrobacterium tumefaciens strains LBA4404 (31) and COR308 (17), provided by Cornell Research Foundation, Inc.…”

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confidence: 99%

A Functional Histidine-Tagged Replication Initiator Protein: Implications for the Study of Single-Stranded DNA Virus Replication In Planta

Vega-Arreguín

Timchenko

Gronenborn³

et al. 2005

J Virol

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Nanoviruses multiply in the nucleus of infected cells by rolling-circle replication (RCR). Upon infection of a host cell, short DNA molecules encapsidated together with the viral ssDNA serve as primers for host polymerase(s) to initiate synthesis of the complementary (minus) strand DNA, creating a double strand (16). The double strand serves as transcription template and for RCR, which is initiated and terminated by replication initiator (Rep) proteins. Nanoviruses encode different Rep proteins; however, only the 33-kDa M-Rep protein is required and sufficient to catalyze replication initiation of its coding DNA and of the other virus genome components (22,39,40). Faba bean necrotic yellows virus (FBNYV) M-Rep, expressed in Escherichia coli, has origin-specific DNA cleavage and nucleotidyl transfer activities in vitro and is an ATPase, both essential functions for viral DNA replication in vivo (39). During RCR the M-Rep protein cleaves the consensus nonamer sequence TAGTATT2AC located at the origin of replication (39), creating a 3Ј-OH terminus and, by analogy to geminivirus replication, is thought to prime viral (plus) strand DNA synthesis (26).For geminivirus Rep proteins, interactions with several host proteins have been described (1, 25). All these different proteins interacting with Rep were identified by using the yeast two-hybrid system. However, due to difficulties encountered in purifying the respective protein complexes, very little is known about the in planta interaction of these proteins. For nanoviruses, nothing is known about the host proteins that interact with M-Rep during replication.We have designed oligohistidine-tagged M-Rep variants of the nanovirus FBNYV that are proficient to catalyze viral DNA replication initiation and termination in Nicotiana benthamiana and report the affinity purification from plant tissue of enzymatically active histidine-tagged M-Rep protein.To the best of our knowledge, this is the first example of a tagged ssDNA virus replication initiator protein that is functional in vivo and that is readily purified from plant tissue. In addition, replicons encoding oligohistidine-tagged M-Rep multiplied and moved systemically along with wild-type FBNYV in its natural host Vicia faba. The replicon encoding the modified M-Rep protein could be transmitted by aphids in a mixed infection with wild-type virus. Finally, we show the in planta interaction of the tagged M-Rep with wild-type M-Rep, suggesting that this protein may be used to identify other protein partners. MATERIALS AND METHODSConstruction of DNA-R-His replicons of FBNYV. Basic molecular biology protocols were as described previously (34). Cloning of FBNYV DNAs of the Egyptian isolate EVI-93 (FBNYV-EG) has also been described elsewhere (39). A BamHI site was introduced by PCR-based QuikChange mutagenesis (Stratagene) at nucleotide position 54 into the noncoding sequence of FBNYV-EG DNA-R (previously designated C2) (23, 39) by using the primers C2BamHI(ϩ)

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Electroporation of megaplasmids into Agrobacterium

Abstract: Received 17 D e c e m b e r 1990; a c c e p t e d 8 J a n u a r y 1991

Cited by 62 publications

References 6 publications

Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures

Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures

Arabidopsis UVH6, a Homolog of Human XPD and Yeast RAD3 DNA Repair Genes, Functions in DNA Repair and Is Essential for Plant Growth

A Functional Histidine-Tagged Replication Initiator Protein: Implications for the Study of Single-Stranded DNA Virus Replication In Planta

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