R 2 represents the coefficient of determination. Intensities of both transcription sites were plotted, with each dot representing a different pair of sister chromatids, n = 240 pairs. ( F) Sister chromatids of the same allele have correlated transcription activity. ( E) Line scan of intensity profiles showing dynamic labeling of DNA and nascent RNAs at corresponding sister chromatids pointed by arrows in (D). ( D) Representative images revealing three distinct transcriptional states of sister chromatid pairs identified by co-labeling of dCas9-GFP 14X and stdMCPtdTomato. ( B, C) Line scan of intensity profiles illustrating dynamic labeling of DNA (green) and nascent RNAs (red) of locus 1 (B) and locus 2 (C) indicated in (A). ( A) Fluorescent images of a cell showing DNA labeling of two LMNA alleles labeled by dCas9-GFP 14X and their transcriptional kinetics indicated by stdMCP-tdTomato at multiple time points. doi: 10.1016/j.jplph.2012.05.026.TriTag monitors allele-specific transcriptional bursting across the cell cycle. GFP-tagging of Arabidopsis acyl-activating enzymes raises the issue of peroxisome-chloroplast import competition versus dual localization. Hooks KB, Turner JE, Graham IA, Runions J, Hooks MA. Remodeling the isoprenoid pathway in tobacco by expressing the cytoplasmic mevalonate pathway in chloroplasts. Kumar S, Hahn FM, Baidoo E, Kahlon TS, Wood DF, McMahan CM, Cornish K, Keasling JD, Daniell H, Whalen MC.

Peroxisomal localization of Arabidopsis isopentenyl diphosphate isomerases suggests that part of the plant isoprenoid mevalonic acid pathway is compartmentalized to peroxisomes. Sapir-Mir M, Mett A, Belausov E, Tal-Meshulam S, Frydman A, Gidoni D, Eyal Y. thaliana chloroplasts leads to growth improvement. Transgenic introduction of a glycolate oxidative cycle into A. Maier A, Fahnenstich H, von-Caemmerer S, Engqvist MKM, Weber APM, Flügge U-I, Maurino VG. Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana. Kebeish R, Niessen M, Thiruveedhi K, Bari R, Hirsch H-JJ, Rosenkranz R, Stäbler N, Schönfeld B, Kreuzaler F, Peterhänsel C. (e, f) Final mRNAs species resulting from alternative splicing of TriTag-1 (e) and TriTag-2 (f). The light tan DNA sequences derive from the TTL 5′ coding region and include sequences encoding the peroxisome targeting sequence (tan). The light green DNA sequences derive from the PIMT2 5′ coding region and include sequences required for chloroplast targeting (green). Donor and acceptor dimers are underlined. Alternatively spliced targeting regions are highlighted. The ATG codon at the end corresponds to the first residue of the GFP open reading frame. (c, d) Design of TriTag-1 (c) and TriTag-2 (d) sequences. (a, b) Splice diagrams of TriTag-1 (a) and TriTag-2 (b), showing non-targeting sequences (gray), chloroplast targeting sequences (light green), peroxisome targeting sequences (tan), and the enhanced GFP coding sequence used in transient expression experiments. This work harnesses alternative splicing and signal embedding for engineering plants to express multi-functional proteins from single genetic constructs.ĭesign of TriTag-1 and TriTag-2, with alternatively spliced chloroplast-targeting PIMT2 and peroxisome-targeting TTL tags.

Our novel signal sequences can reduce the number of cloning steps and the amount of genetic material required to target a heterologous protein to multiple locations in plant cells.

TriTag-3 embeds a conserved peroxisomal targeting signal within a chloroplast transit peptide, directing GFP to the chloroplasts and peroxisomes. TriTag-1 shows a bias for targeting the chloroplast envelope while TriTag-2 preferentially targets the peroxisomes. TriTag-1 and TriTag-2 use alternative splicing to generate differentially localized GFP isoforms, localizing it to the chloroplasts, peroxisomes and cytosol. We designed novel hybrid signal sequences for multiple-compartment localization and characterize their function when fused to GFP in Nicotiana benthamiana leaf tissue. Plant bioengineers require simple genetic devices for predictable localization of heterologous proteins to multiple subcellular compartments.

Tritag pyramid