Inheritance and structure of foreign DNA in progenies of transgenic tobacco obtained by direct gene transfer

Summary One of the transformed tobacco plants obtained by direct DNA transformation possessed two marker genes, a chimeric aminoglycoside phosphotransferase and nopaline synthase genes. Selfed progenies of this plant (T3-d) showed stable inheritance of these two genes. The minimum size of foreign DNA integrated into tobacco genome was estimated to be 5.4 kbp. A deleted nopaline synthase gene co-existed with an intact gene. The linkage analysis indicated that two transformants, T1-b and T3-c, possessed foreign DNA inserted in different chromosomes or in different sites of the same chromosome that recombine freely.     

Evidence for the location of foreign genes in three different chromosomes in a plant obtained from direct DNA transfer

Tobacco mesophyll protoplasts were treated with plasmids, pCT2 (17.1 kbp) or pCT2T3 (18.3 kbp), which contained a chimeric aminoglycoside phosphotransferase II (APH(3′)II) gene and an intact nopaline synthase gene. Expression of two marker enzymes, APH(3′)II and nopaline synthase, were analyzed in transformed plants. Four out of 16 transformants obtained by pCT2T3 possessed both enzymes. Upon self-pollination, the progeny of one of transformants (T2) segregated to 153∶4 in terms of resistant and susceptible character to kanamycin, suggesting insertion of foreign genes into three independent chromosomes. The kanamycin resistant character in the rest of transformants showed 3∶1 segregation. DNA blot analysis of the T2 transformant and progenies indicated the presence of two marker genes.     

In Vitro Binding of Wheat-Germ Proteins to the 5'-Upstream Region of the rolC Gene of Ri Plasmid

In vitro binding of nuclear proteins from wheat germ to the5'-upstream region of the rolC gene of Ri plasmid was investigated.The specific DNA sequences interacting with proteins were detectedby DNase I footprinting. (Received October 8, 1990; Accepted November 30, 1990)     

Conditions Favorable for the Somatic Embryogenesis in Carrot Cell Culture Enhance Expression of the roIC Promoter-GUS Fusion Gene

We obtained carrot (Daucus carota) cells possessing the 5′-noncoding sequence of the ORF12 gene (roIC) of TL-DNA of the Ri plasmid and a structural gene of bacterial β-glucuronidase by Agrobacterium-mediated transformation. When such cells were cultured in medium containing 2,4-dichlorophenoxyacetic acid, substantial reduction in β-glucuronidase activity was observed. Upon transferring the cells from a 2,4-D-containing medium to one devoid of 2,4-dichlorophenoxyacetic acid, enhanced expression of β-glucuronidase in somatic embryo development was recorded. Activation by gibberillic acid and suppression by abscisic acid of β-glucuronidase activities, in concord with embryogenesis, were also noted.     

Uptake of liposome-encapsulating plasmid DNA by plant protoplasts and molecular fate of foreign DNA

Summary Conditions faborable for the uptake of artificial lipid vesicles (liposomes) encapsulating plasmid DNA byDaucus carota protoplasts were investigated. Incubation period necessary for the maximum uptake of liposome-DNA was in the neighborhood of 10 minutes under the circumstances where liposomes (approximately 1.28 moles lecithin/ml) were mixed with 5 × 10⁶ protoplasts/ml. Results obtained by Southern hybridization indicated that the recombinant DNA vector, pBR325 was found in protoplasts after 20 hours incubation in the open circular, linear and some complexed forms. There were some variations in DNA-uptake among protoplasts from different plant species. The gradual disappearance of plasmid molecules was confirmed after 1 week culture, suggesting instability of pBR325 in prolonged cell culture.     

Non-systemic expression of a stress-responsive maize polyubiquitin gene (Ubi-1) in transgenic rice plants

We have used the promoter, 1st exon and 1st intron of the maize polyubiquitin gene (Ubi-1) for rice transformation experiments and revealed the characteristic expression of Ubi-1 gene: (1) Ubi-1 gene is not regulated systemically but rather individual cells respond independently to the heat or physical stress; (2) Ubi-1 gene changes its tissue-specific expression in response to stress treatment; (3) the expression of Ubi-1 gene is dependent on cell cycle.     

Analysis of randomly isolated cDNAs from developing endosperm of rice (Oryza sativa L.): evaluation of expressed sequence tags,and expression levels of mRNAs

Using a cDNA library prepared from poly(A)⁺ RNA from 10-day-old rice endosperm, partial nucleotide sequences of randomly isolated clones were analyzed. A total of 153 (30.6%) out of 500 cDNA clones showed high amino acid identity to previously identified genes. There was significant redundancy in cDNAs encoding prolamine and glutelin. About 21.0% of the cDNA clones were found to code for seed storage protein genes. Consequently, 37 independent genes were identified. Using cDNA clones encoding glutelin, prolamine, seed allergen, -1,4-glucan branching enzyme, glycine-rich RNA binding protein, metallothionein, non-specific lipid-transfer protein and ubiquitin conjugating enzyme the accumulation of mRNA during rice seed development was compared. Genes associated with seed storage protein and starch biosynthesis were expressed according to expected developmental stages. Glycinerich RNA binding protein genes as well as metallothionein-like protein genes were highly expressed in developing seeds, but low in leaves of whole plants.     

Quantitative analysis of the fate of exogenous DNA in Nicotiana protoplasts

After a 5-hour incubation of protoplasts of Nicotiana tabacum L. ;Xanthi' with (3)H-DNA (7.26 mug/ml) from N. tabacum L. ;Xanthi nc' 3.5% of the initial radioactivity was found in acid-insoluble substances of the protoplasts. The addition of DEAE-dextran and poly-l-lysine to the incubation medium nearly doubled radioactivity adsorption. The absorption was inhibited by 2,4-dinitrophenol, KCN, and low temperature (0 C); this inhibition could not be reversed by exogenous ATP. About 500 tobacco plants established from protoplasts of a normally tobacco-mosaic virus-susceptible cultivar that had been allowed to absorb DNA prepared from a resistant cultivar did not show transfer of the virus-resistant gene.A detailed analysis was performed of the disposition of exogenous DNA in plant protoplasts, by employing Escherichia coli(3)H-DNA and Nicotiana glutinosa protoplasts. In 5 to 20 hours, about 10% of the (3)H-DNA entered the protoplasts. Competition experiments between the (3)H-DNA and unlabeled DNA or thymidine showed that the entry occurred as undegraded (3)H-DNA. Examination of intraprotoplast fractions revealed that 60 to 80% of the absorbed radioactivity resided in the "soluble" fraction of the cytoplasm and 20% in the nuclear fraction. The mitochondrion fraction also contained measurable radioactivity. Sizing on sucrose density gradients showed that the bulk of the absorbed E. coli DNA had been depolymerized. Of the incorporated radioactivity, 15% was accountable as DNA, exogenous as well as resynthesized, and 15% as RNA, protein, and other cell constituents. DNA/DNA hybridization test indicated that 17.6% of the re-extractable (3)H-DNA retained homology with the E. coli DNA; this was equivalent to 2.6% of the absorbed radioactivity. Resynthesized receptor protoplast DNA was represented by a fraction at least 1.7% of the total absorbed radioactivity. The amount of bacterial DNA remaining in protoplasts suggests that each protoplast retained 2.3 x 10(-15)g donor DNA, or approximately half of the E. coli genome.