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Summary A 12.5-kb DNA fragment with junction regions between the transgene and genomic DNA was cloned from a transgenic tobacco cell line obtained by microprojectile bombardment of plasmid pCaMVNEO. Nucleotide sequence analysis of the fragment (DDBJ accession no. D84238) showed that it carried a 7.7-kb core sequence (concatemer of a complete pCaMVNEO and a partial pCaMVNEO) and two identical 1.3-kb junction sequences that flanked both the 5′ and 3′ ends of the core sequence and had inverted orientations. These sequences had topoisomerase II (Topo II) cleavage sites and adenine and thimine-rich sequences known to be specific to nuclear scaffold-attachment regions (SARs). An in vitro binding assay showed that a 507-bp fragment (designated TJ1) from the 1.3-kb sequence had the ability to bind to nuclear scaffold preparations of cultured tobacco cells, confirmation that the 1.3-kb sequence is an SAR. Insertion of TJ1at the 5′ and 3′ sides of the expression cassette for the npt II gene increased transformant yields 5- to 10-fold and the NPT II enzyme activity per copy of the gene 5-fold. TJ1enhances the integration or expression of the transgene, or both. Clearly, TJ1is very useful for producing transgenic plants. This is the first report on an SAR-like sequence that is located in the transgene locus and enhances transformation efficiency in eukaryotic cells. The possible role of TJ1-SAR in the molecular evolution of plant genome is discussed. [ABSTRACT FROM AUTHOR]

Summary: The emission of N[sub 2] and N[sub 2]O from intact transgenic tobacco (clone 271) expressing antisense nitrite reductase (NiR) mRNA, and wild-type plants grown aseptically, on NO[sub 3][sup –], NO[sub 2][sup –] or NH[sub 4][sup +] -containing medium was investigated. [sup 15]N contents of gas sampled from gas-sealed pots, in which the plants were grown on [sup 15]N-containing medium, were analyzed by gas chromato- graphy and mass spectrometry (GC–MS). No emission of N[sub 2] was detected in either of the gas samples from plant clone 271 or the wild-type grown on NO[sub 3][sup –]-containing medium. N[sub 2]O emission from clone 271 grown on NO[sub 3][sup –]-containing medium was detected, but not from the wild-type plants. The N[sub 2]O emission rate of clone 271 was 106 ng N[sub 2]O mg[sup –1] incorporated N week[sup –1] and the N[sub 2]O emission was inhibited by tungstate (a nitrate reductase inhibitor). No emission of N[sub 2]O was found from clone 271 or wild-type plants grown on medium containing NH[sub 4][sup +]. Emission of N[sub 2]O also was detected from clone 271 grown on NO[sub 2][sup –]-containing medium and its emission rate increased with increasing NO[sub 2][sup –] levels in plants. We speculate that NO[sub 3][sup –] is reduced to NO[sub 2][sup –] and that a part of NO[sub 2][sup –] is metabolized to N[sub 2]O in clone 271. [ABSTRACT FROM AUTHOR]

Tobacco (Nicotiana tabacum L. cv. Xanthi XHFD8) possesses four nitrite reductase (NiR) genes: nii1, nii2, nii3, and nii4. Their differential expression in leaves and roots was investigated by quantitative competitive RT-PCR using gene-specific primer pairs. These results appear to contradict existing views on the expression of these NiR genes: (i) the mRNA of each of the four NiR genes was distinguishable both in leaves and roots; (ii) nitrate treatment increased nii1 and nii3 mRNA in leaves and roots by at least 4-fold (at least 5-fold in nii2 and nii4 mRNA); and (iii) the steady-state levels of nii1 and nii3 mRNA were almost the same in leaves (6–7×105 and about 3×106 copies μg−1 of total RNA before and after nitrate treatment, respectively) and in roots (3–4×104 and 3–6×105 copies μg−1 of total RNA before and after nitrate treatment, respectively). Very similar relationships were obtained for the steady-state levels of nii2 and nii4 mRNA in roots (2–4×105 and 8×106 copies μg−1 of total RNA before and after nitrate treatment, respectively), and in leaves (5–9×104 and 4×105 copies μg−1 of total RNA before and after nitrate treatment, respectively). These results demonstrate that nii1 and nii3 transcripts are a dominating, but not exclusive, NiR mRNA in leaves, and the same is true for nii2 and nii4 transcripts in roots. [ABSTRACT FROM PUBLISHER]

Levels of NO-3, NO-2 and NH+4 ions in leaves of six plant species were determined by capillary electrophoresis. Levels of NO-3 ions differed by a factor of more than 350 in the six species. NO-2 and NH+4 ions were detected in all species examined but at lower and more similar respective levels than NO-3 ions. [ABSTRACT FROM AUTHOR]

Indicates that all correspondence concerning 'The Plant Journal' paper titled 'Emission of nitrous oxide (N[sub 2]O) from transgenic tobacco expressing antisense NiR mRNA' should be addressed to Hiromichi Morikawa, Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.