1724 The Plant gene organization event. All seven genes have they are - PDF document

1724 The Plant gene organization event. All seven genes have they are not organized a repetitive cluster, as are genes (Beier Apart from RNA polymerase terminator element and Brown, recognizable conserved flanking sequence that sug- sequence element whether these genes are expressed enable studies their transcription having the anticodon and thereby them able press reporter genes nonsense codon. of the tRNA Gene a method plant tRNA vivo that relies translational suppression nonsense codons the chloramphenicol acetyltransferase reporter gene (Franklin firefly luciferase sensitive reporter plants encouraged sense suppression (Schultz and Luehrsen et that end, from the Arabidopsis (Ecotype Columbia) Each gene with nucleotide in bold capital termination signals tRNATrp6, and optimize alignment. Translational Suppression sient expression experiments. ochre, and in place carrot protoplasts with vary- circles, line with the point represents the three inde- cauliflower mosaic virus (CaMV) tobacco mosaic encode a ochre (oc; UAA), stop codons (Figure The fourth gene was site for demonstrated previously that N-terminal residues for activity DNAs encoding LUCoc, or into carrot protoplasts, when plasmids containing express a with a codon were activity reached 2 the background lumines- alone (Figure assay for the LUC more sensitive; furthermore, poly- ethylene glycol (PEG)-mediated transformation (rather described) required DNA. These greatly improved our ability to tRNA gene genes from Arabidopsis no obvious sequence homologies either the 3‘flanks, apart the putative terminator determine whether these whether significantly flanking these genes, four code amber suppressors protoplasts together with Measuring the suppression indicated all expressed, small variation suggests is unlikely that flanking sequence the sequences wholly contained within the fragment genomic DNA present quences and B’sequences), constructs or 24 ing sequences. The observed suppression increased proportionally with increasing molar amounts plasmid with copies, representing a fourfold the single-copy con- approximately fourfold more the single-copy construct (Figure 38). This is expressed independently. construct containing 24 copies, only a small increase the construct contain- copies, suggesting system is saturable. This might occur processing, aminoacylation, upstream regions be expressed together with the tRNALeU gene shown be expressed an Arabidopsis and shown be expressed (data not shown), that all region (Figure Because the is required to determine to these the plant footprint analyses the noncoding and the coding DNA both before incubation with recombinant from Arabidopsis. These analyses demonstrated between nucleotides +1 and (centered around -26) (Figure The observed the 5’flanking whether the the natural 5’flanking E E E Nonsense Suppression among Relative efficiencies nonsense suppression genes. Carrot protoplasts transfected with reporter plasmid together with (40 pg) constant each experiment. activity relative reporter construct efficiency variability. Error bars the standard deviation the tRNA2tz gene on expression. Carrot protoplasts plasmid along with 5 (40 pg) constant each experiment. represented as activity relative the wild-type reporter construct each transformation as efficiency variability. Error bars resent the standard deviation Gene Expression 1727 Sequences on Nucleotide sequences tRNA$&l mutants, The sequence the native shown on are deletion -1 and these mutants with the the vector the early mutant. The A-1EPrev five point the natural oligonucleotide (stippled containing the twodifferent positions from these A-1TATA_3z, A-10TATA-3z, A-1TATA-z4. contains two Carrot protoplasts cotransfected with region. All activity relative to the pair obtained with the protoplasts to The 8-glucuronidase reporter construct to correct for transfection efficiency variability. bars represent flanking region resulting construct these point mutations dramatic, because gene contains the region define whether a single element important, a element from the adenovirus 2 major late promoter was nucleotide -32. This increased expression approximately position is important modulatory element. is likely that either additional elements are required maximal expression the position the newly introduced the position sequences had a surprisingly strong start site profoundly reduced suppression, and was not the natural sequences (compare with A-lTATA-32 Figure 6B). analyze the RNA products these genes carrot protoplasts primer extension with a primer ending the unique anticodon but successful because the transcriptase encountered a major stop and generated truncated products not shown). the published sequence and Ghosh, a 2,2-N-dimethylguanine, which is likely block the transcriptase. This problem, together with a very the deletion mutants prevented us from mapping the transcription constructs expressed these constructs with a heterologous extract 293 cells the wild-type the deletion and the constructs with the yielded transcripts indicating that lanes 3 the constructs with the yielded shortened be initiated within the coding sequences. Such gin would explain these constructs lanes 8 and carried out a primer extension analysis in vitro transcripts analysis indicated and the constructs be- the position the mature tRNA; Figure lanes 2 and whereas transcription and the A-lTATA-32 con- begins predominantly position -1 (Figure 3 and 1728 The -51 -41 -31 -21 -11 -1 10 -51 -41 -31 -21 -11 -1 10 Relative Posiiion -51 -41 -31 -21 -11 -1 10 -51 -41 -31 -31 -21 -11 -1 10 -51 -41 -31 -21 -1 10 -51 -41 -31 -21 -11 -51 -41 -21 -11 -1 10 Relative Posiiion helical stability genes and for 5‘ flanking sequences using the Thermodyne program described with the default Na+. The window size used 5' flank- not completely transcription completely may suboptimal localization the mammalian additional sequences. have recently suggested that sequences immediately downstream or subsequent plant tRNA these plant tRNA suggests that the mammalian apparatus might despite these elements not mammalian tRNA relative contributions promoter elements strongly influenced elements appear transcription efficiency to initiation site selection by Sandmeyer, 1993; both the suggest that There may alternative forms with the and the other with with both the coordinate interactions suppressors that function tRNA genes the synthesis insights into elements affecting control the expression and Sequence Analysis Arabidopsis thaliana ecotype EMBL3 genomic Klee, Monsanto Corp., MO) was probe containing the sequence and prehybridized buffer containing a 350-bp product containing the DNA by washed once with 2 sodium citrate) buffer containing then twice phage isolates with the Specific primers to the opposite orientations primers specific sequence cod- into the yielding constructs desig- Genes and Mutant Luciferase Genes (complementary with the stop codon). Muta- performed using and trimolecular specific primers with a 5' end codon and nucleotide), and the and forward primers, to prepare thus introduc- specific mutation(s). with a enzyme from the polylinker DNA was digested with the ligated with ligation reaction. used for the plasmid KS+, with the 350-bp the presence polyethylene glycol. codons, the plasmid pGEM-luc the cauliflower and the nopaline EcoRl frag- containing the site), and EcoRl site pBluescriptll KS-. plasmid (pKS[-]35SE) mutagenized using Nonsense mutants fused to the trimolecular ligation and a Ncol-Spll-digested mutations and the Spll-Sal1 gene fragment from pGEM- All mutations sequencing using Transformation of Carrot Protoplasts Carrot (Daucus carota) described previ- into carrot protoplasts The Plant Cell modifications; the from a 40-mL suspension protoplasts. Protoplasts and resuspended and immediately before trans- formation, the protoplasts mM Mes, 20 mM mannitol, pH alkaline lysis and purified through CsCl room temperature lution (40 70 mL solution containing mannitol; the solution stored frozen mixed and incubated room tempera- Murashige and Skoog thiamine, pyridoxine, nicotinic acid, mg/L glycine, room tempera- Carrot protoplasts cell cul- ture lysis reagent (Promega). From 2 cell extract room temperature assay reagent (Promega). a luminometer (model3010; Analytical Sci- entific Instruments, and counted with the wild-type that the assays remained P-Glucuronidase assays were the 5‘flanking region into the KS+. The lacks the natural (including the termination site) downstream from the coding region. product from the Sall-Hindlll-digested plas- The sequences mutants were for footprinting on the coding and noncoding isolating the and Hindlll-Sacl fragments from and end-labeled Klenow fragment (TBP) (At2; Rockefeller University, New York, from Escherichia aglutathione S-transferasefusion (construct Missouri-Columbia) and cleaved the glutathione S-transferase tag. TBP TBP dC], 10 pg of BSA, 15 mM ‘Hepes, pH 7.9, 50 mM KCI, 6 mM MgCI2, glycerol) for room tempera- Then. assay each sample up to 200 (25 ng) added to each sample, and digestion room temperature The reaction solution (0.2 and loaded onto a were de- overnight exposure at -70% Vitro Transcription Deletion Mutants the deletion mu- lacking the natural S’flanking and including The A-1TATA_32 an annealed double-stranded adenovirus 2 major the Notl and and A-IO+TATA_24 mutants were constructed constructs, respectively. either orientation the for- reverse orientations, respectively) constructed by oligonucleotide containing the early simian virus into the the coding assays were performed using reactions containing The products were analyzed Primer Extension Analysis radiolabeled with and used the human with Tth thank Tim Ulmasov Alexander Kenzior contributing sug- Sarah Scanlon tRNATrp Gene Expression 1733 constructing the fusion protein, and David Thermodyn program. work was the Universityof Missouri for the Received June accepted August Allison, D.S., the 3'flank- sequences on Variable patterns leaves. Proc. Cell. Biol. are highly amplified Arabidopsis. MOI. Gen. Genet. 225, 72-80. D.F., and Nucleotide sequences transcription termination. J., and gene expression plant cells utilizing antisense Marsolier, M.C., Basal promoter enhancer elements 233, 644-658. Raymond, G.J., gene expression tRNA mediated sup- plants. Plant Acad. Sci. USA90, cells. Cell 69, 685-696. Eschenlauer, J.B., Kaiser, M.W., Cell. Biol. Feinberg, A.P., and Vogelstein, endonuclease fragments specific activity. Roeder, R.G., and Arabidopsis contains two genes Whitehall, S.K., Geiduschek, Cell. Biol. Structure and function from wheat germ. Acids Res. and Filipowicz, pre-mRNA processing transfected plant protoplasts. Methods En- 181, 148-161. Jefferson, R.A. chimeric genes plants: The and Blanco, and function yeast RNA and Kowalski, (1994). DNA instability facilitates replication origin. and Folk, genes from Arabidopsis. Plant MOI. Luehrsen, K.R., deWet, J., and (1992). Transient sion analysis plants using firefly luciferase reporter Weil, J.H., and Dietrich, Plant Physiol. Plant Dujardin, G., Gerard, J., and tein factor sequence requirement for transcription vitro. Plant Natale, D.A., Schubert, A.E., and Kowalski, (1992). DNA mutational defects a yeast Proc. Natl. USA 89, Hale, C., tRNAAla gene upstream sequence elements. Nucleic Acids a nuclear tRNAMe" gene from soybean. G.J., and and processing yeast tRNA. Acids Res. J., Fritsch, E.F., and Laboratory Manual, 2nd ed. (Cold Spring Harbor, NY: and Folk, minichromosomes. Nu- Acids Res. tRNA-mediated nonsense suppression. Plant Cell and Olson, quences on RajBhandary, eds (Washington, DC: American and Capone, vitro transcription tRNA gene. Nucleic Acids and Gesteland, soybean genes from tRNAASp and MOI. Appl. by RNA from wheat Transcription. (Austin, (1992). A role for the factor IID (1993). RNA and Berk, transcription factor Proc. Natl. 1723-1734, October American Society Plant Physiologists Barbara Ulmasov and William Biochemistry, University Columbia, Missouri Arabidopsis and studied the initiation t 1 1 a a I a a • a T C a a a a + a sequences, a a