Yeast RAS2 affects cell viability, mitotic division and transient gene expression in Nicotiana species

Hilson, Pierre; Dewulf, Jocelyne; Delporte, Fabienne; Installé, P.; Jacquemin, Jean-Marie; Jacobs, M.; Negrutiu, Ioan

doi:10.1007/bf00016500

Cited by 25 publications

(11 citation statements)

References 38 publications

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“…Plasmid constructs pGP6 and pHB2 contain the npt from Tn5 (conferring kanamycin resistance) with the CaMV (cauliflower mosaic virus) 35S promoter in a pUC18 plasmid (Hilson et al, 1990;Negrutiu et al, 1990). pGP6 is sketched in Fig.…”

Section: Protoplast Isolation and Transformationmentioning

confidence: 99%

Expression instability and genetic disorders in transgenicNicotiana plumbaginifolia L. plants

Cherdshewasart¹,

Gharti-Chhetri²,

Saul

et al. 1993

Transgenic Research

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The ease of integrative transformation with foreign genes and the extent of their expression and stability in successive generations determine the applicability of direct gene transfer. In Nicotiana plumbaginifolia, one to ten copies of foreign DNA were integrated into the plant genome, resulting in simple to complex patterns of integration. Genetic analysis showed that in more than 50% of the cases, this DNA inserted at two or more loci in the genome. Of the 156 crosses performed between F1 monogenic transformants, only eight combinations showed linkage of the inserted neomycin phosphotransferase genes (npt). The following instability events were registered: physical loss, alterations in the initial segregation rates in successive meiotic generations observed in either selfing or crossing (reduction or increase in number of segregating loci) and genomic disorders in crosses between transformants. Among them of particular interest were the "discordant" segregation values observed between corresponding R~ and F~ progenies in up to 9% of the evaluated transformants. In addition, 5 % of the transformants showed a phenotypic loss of resistance. In the F 3 generation, 5 out of 15 transformants exhibited instability, which was transmitted to the F4 generation. Further increases in instability rates were observed with higher numbers of insertion loci and in crosses between independent transgenic plants, reaching 100% when a trigenic partner was involved. N. plumbaginifolia exhibited more instability than N. tabacum under equivalent experimental conditions. The molecular bases of such instability events are discussed in relation to DNA methylation, co-suppression and genomic imbalance.

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Section: Protoplast Isolation and Transformationmentioning

confidence: 99%

Expression instability and genetic disorders in transgenicNicotiana plumbaginifolia L. plants

Cherdshewasart¹,

Gharti-Chhetri²,

Saul

et al. 1993

Transgenic Research

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“…1) is the toxicity is localized to specific cells because the gene product is an intracellular toxin which is not active once outside the cell. A chimeric RAS2 gene from yeast also had a "killer" effect in Nicotianaplumbaginifolia; however, the manifestation of toxicity was species-specific and depended on other conditions (16).…”

Section: Construction Of Promoter-probe Vectormentioning

confidence: 99%

Expression of DNA Coding for Diphtheria Toxin Chain A Is Toxic to Plant Cells

Czakó

An²

1991

Plant Physiol.

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DNA coding for the enzymatically active subunit A of diphtheria toxin was placed under the control of the cauliflower mosaic virus 35S promoter and the Agrobacterium left transfer-DNA gene 7 polyadenylation signal. Agrobacteria carrying a binary plant vector with the chimeric diphtheria toxin A gene had very low transforming activity in tobacco (Nicotiana tabacum L.), and greatly diminished the recovery of stable transformants when mixed together with agrobacteria which alone transformed plant cells well. As reporter genes, the bacterial cat2 and fl-glucuronidase genes have been used most widely (14,19). Both gene activities can be quantified, and the latter can be detected in situ; however, the assays are still tedious and destruct the material. In addition, they are sometimes complicated by endogenous inhibitors or background activity (10,15,19 (8), and the active site is distinct from that for the ADP-ribosylation (6). The structural gene for DTx carried by corynebacteriophages codes for a protein with a leader peptide which is removed during secretion from Corynebacterium diphtheriae. The mature toxin is composed of two domains: the NH2-terminal portion (A chain) which carries the active sites of the toxin and the COOH-terminal portion (B chain) which is required for binding to specific receptors, transport of the toxin into target cells, and is dispensable for intracellular toxicity. The toxin protein is unmasked and activated by reduction of the disulfide linkages and by hydrolysis of a peptide bond located in the loop between the sulfur atoms of the disulfide bridge located in the NH2-terminal portion of the intact toxin.The two fragments remain firmly held together by weak interactions (27).Because polypeptide fragments of microbial and plant toxins (including DTx) that are devoid of cell receptor binding and transfer domains are not cytotoxic, it has become attractive to use these polypeptides in yeast (29) and mammalian (21, 26) systems in the assembly of chimeric genes. Here we present evidence that expression of a chimeric DTx-A gene construct in tobacco is toxic. This finding provides a potent new negative selection gene for probing the function of plant promoters. MATERIALS AND METHODS Strains and Molecular CloningEscherichia coli strain MC 1000 (7) was used as a recipient for plasmids constructed by standard procedures. The Agrobacterium tumefaciens strain LBA4404 (17) carrying the pAL4404 disarmed helper Ti plasmid in a Ach5 chromosomal background was used for plant transformation. The DNA coding for the DTx fragment A was obtained from pTH 1 (21). The binary plant expression vectors pGA642 and pGA643, carrying a polylinker site between the nopaline synthase promoter and CaMV 35S promoter, respectively, and the pTiA6 octopine-type Ti-plasmid TL-DNA 7 gene terminator were described previously (4). The binary vectors were transferred into Agrobacterium by the direct transformation method (2).

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“…Mammalian ras gene products are involved in the control of ceil growth and divisions. In Nicotiana plumbaginifoii'a ceils, overexpression of the Saccharomyces cerevisiae ~qAS2 gene inhibits cell viability and mitotic division [3]. The products of the rho gene family are involved in cytoskeletal organization [4,5].…”

Section: Introductionmentioning

confidence: 99%

In vitro mutation analysis of Arabidopsis thaliana small GTP‐binding proteins and detection of GAP‐like activities in plant cells

Anai

Matsui²,

Nomura³

et al. 1994

FEBS Letters

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Absia'ae¢Previously, we have reported the molecular cloning of ara genes encoding a small GTP-binding protein from ArabMopsis thaliana. The criterion based on amino acid sequences suggest that such an ara gene family can be classified to be of the YPTlrab type. To examine the biochemical properties of ARA proteins, several deletions and point mutations were introduced into ara cDNAs. Mutant proteins were expressed in E. coIi as GST-chimeric molecuies and analyzed in terms of their GTP-binding or GTP-hydrolysing ability in vitro. The resuits indicate that ~ur conserved amino acid sequence regions of ARA proteins are necessary for GTP-binding. A point mutation of Ash at position 72 for ARA-2, or 7i for ARA-4, to Ile decreased GTP-binding and a point mutation of Gln at position 126 for ARA-2, or 125 for ARA-4, to Leu suppressed GTP-hydrolysis activity. Furthe~ore, certain factors associated with the membrane fraction accelerated GTPase activities of ARA proteins, suggesting the presence of GTPase activating protein(s) (GAP(s)) in the vesicular transport system of higher plant cells.

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Yeast RAS2 affects cell viability, mitotic division and transient gene expression in Nicotiana species

Cited by 25 publications

References 38 publications

Expression instability and genetic disorders in transgenicNicotiana plumbaginifolia L. plants

Expression instability and genetic disorders in transgenicNicotiana plumbaginifolia L. plants

Expression of DNA Coding for Diphtheria Toxin Chain A Is Toxic to Plant Cells

In vitro mutation analysis of Arabidopsis thaliana small GTP‐binding proteins and detection of GAP‐like activities in plant cells

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