Effects of rho‐independent terminators on the expression of the prokaryotic β‐glucuronidase gene in tobacco

Abstract

The threonine (thr) attenuator with a dyad symmetrical struc-uridine residues were also analyzed, and the results suggested ture is from the regulatory region of the thr operon of that the variants with the shorter uridine stretch produced more GUS protein than those with the longer stretch. Trans-Escherichia coli, and encodes RNA with a stem-and-loop structure followed by a stretch of uridine residues. The thr genic tobacco plants with the thr attenuator and its variants located between the 35S promoter and GUS coding region attenuator and its variants were subcloned into the region between the 35S promoter and -glucuronidase (GUS) coding were also generated, and their steady state RNAs were hybridized with 2 radioactive antisense RNA probes which region, and the transient expression of GUS gene in tobacco protoplast was treated as a reporter for gene regulation in bound 5% and 3% of the thr hairpin, respectively. After the plants. Results from the 14 variants in the stem region of the digestion of S1 nuclease, the amount of the nuclease-resistant transcript from the protection of the 5% antisense RNA probe thr attenuator indicated that both base pairing and sequence specificity in the G + C-rich region of the stem were important was much more than that from the protection of the 3% probe in all tested variants. This result suggests that these dyad for the GUS expression, but 1 base mismatch in the A+ Usymmetries may affect transcription of plant RNA polymerase rich region of stem did not affect the GUS expression in plants. Seven variants with nested deletion in the stretch of II.

Effects of the multiple polyadenylation signal AAUAAA on mRNA 3′-end formation and gene expression

“…The fluorescence of the samples was measured by the excitation wavelength at 365 nm and the emission wavelength at 455 nm, using a Hitachi Spectrofluorometer F2000. A solution of 100 mM 4-methyl umbelliferone (MU) in 0.2 M sodium carbonate was used to calibrate the intensity of fluorescence (Jefferson et al 1987;Jeng and Yen 2000;Chu and Jeng 2002). The concentration of protein in each cell extract was measured by Coomassie Blue Binding method (Sedmak and Grossberg 1977;Spector 1978).…”

“…Plasmid MTC301 containing the 35S promoter and firefly luciferase was kindly provided by Dr. Su-May Yu (Institute of Molecular Biology, Academia Sinica, Taiwan), and was treated as an internal control during electroporation to compare quantitatively the GUS expression from different constructs. The electroporation procedure was reported (Jeng and Yen 2000;Chu and Jeng 2002) and modified as follows. Protoplast isolated from tobacco leaves was adjusted by a dilution solution (0.4 M D-mannitol, 8 mM MES-KOH, and 5 mM CaCl 2 ) to obtain 2 9 10 6 cell/ml.…”

“…The 3 0 -UTR whose transcript contains two canonical AAUAAA signals from endochitinase gene was isolated from tobacco genome and replaced the NOS terminator in the pBI221SI (Jeng and Yen 2000;Chu and Jeng 2002) to become pAT with GUS gene as reporter (Fig. 1).…”

“…After digested by SacI and EcoRI, this 403-bp fragment was cloned into the corresponding sites of pBI221SI (Jeng and Yen 2000) to become pAT with GUS gene followed by 3 0 -UTR from endochitinase gene (Fig. 1).…”

Multiple transduction pathways regulate the 35S promoter with an ABA responsive element

“…Schematic representation of the construction of plasmids 1R, 2R, 1L and 2L. The CaMV 35S promoter in pBI221SI [21] was divided into two parts. One 723-bp fragment, the 5?…”