Rekombinantní GFP technologie a jejich využitícr-hana.upol.cz/cellbiol/bilder/GFPT/GFPT5.pdfefficient translation of the transcribed gene in E. coli 5. The sequence AACA was inserted

Rekombinantní GFP technologie a jejich využití

Využití technologie fluorescenčních proteinů u rostlin

Specifické tagování fluorescenčních proteinů pro

dynamické studium buněčných organel

GFP - a fully genetically encoded label

- can be directly visualized

- allows the direct visualization of gene expression and subcellular localization

of fusion proteins, in living cells, without the need for invasive techniques or addition of

cofactors

the chromophore group - the tripeptide within the protein is cyclized and

oxidized to form a covalently attached chromophore

- is formed by a unique posttranslational

modification of the three amino acid residues of the

helix at positions 65–67, and other residues of the

molecule

•First experiments in plants (1995)

- GFP has been successfully expressed at

high levels in tobacco plants using the cytoplasmic RNA

viruses potato virus X and tobacco mosaic virus.

- In these experiments, the gene was

directly expressed as a viral mRNA in infected cells, and very

high levels of GFP fluorescence were seen.

- However, poor or no fluorescence was

seen when the gfp cDNA was transformed into isolated cells

or transformed plants of Arabidopsis.

Construction of a gfp Expression Cassette for Arabidopsis Haseloff et al. (1997) :

1. A synthetic gfp gene was constructed using PCR

2. The plasmid pGFP10.1, which contains a cloned A. victoria gfp cDNA sequence, was

used as template for amplification, and synthetic oligomer primers were used to

modify the sequences flanking the GFP coding region.

3. Recognition sites for the restriction endonucleases BamHI and SacI were placed at

the 59 and 39 termini of the amplified fragment

4. A Shine-Dalgarno sequence was positioned upstream of the initiation codon to ensure

efficient translation of the transcribed gene in E. coli

5. The sequence AACA was inserted between positions 24 and 21 for efficient

translation in plants

6. Expression of the gene cassette in transformed E. coli and yeast cells gave rise to

brilliant green fluorescence under long-wavelength UV illumination.

- They determined that the PCR-amplified gfp cDNA cassette correctly produced

fluorescent protein product in transformed microbial cells.

- The sequence was inserted into the plant transformation vector pBI121, replacing the

GUS gene behind the CaMV 35S promoter.

- An Agrobacterium mediated root transformation procedure of Arabidopsis was used,

but no GFP-related fluorescence have been observed…

Haseloff J, Siemering RK, Prasher DC, Hodge S. 1997. Removal of a cryptic intron and subcellular localization

of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proceedings of the

National Academy of Sciences, USA 94, 2122-2127.

Haseloff et al. (1997) have found that proper expression of GFP is

curtailed due to aberrant mRNA processing (exprese GFP cDNA v

buňkách Arabidopsis je omezena abnormálním sestřihem mRNA).

gfp mRNA Is Misspliced in Arabidopsis Haseloff et al. (1997) :

1. They used PCR-based methods to verify the correct insertion of the 35S promoter-

driven gfp cDNA and to check mRNA transcription and processing in transformed

plantlets.

2. The gfp sequences in extracts were therefore derived from genomic DNA or

transcribed mRNAs, respectively. The gfp sequences were amplified via PCR from

these separate extracts, and the products were analyzed for the presence of certain

restriction endonuclease sites.

3. While the expected full-length product was obtained after amplification of the

integrated gfp gene, RT-PCR of gfp mRNA sequences gave rise to a truncated

product.

4. This product was 80–90 bp shorter than expected and was missing recognition sites

for the restriction endonucleases DraI and AccI.

5. This is consistent with a small deletion within the coding sequence of the gfp mRNA.

6. Because the amplified gfp gene sequences were of the expected size, they

presumed that the mRNA was misprocessed.

Aberrant posttranscriptional processing of gfp mRNA. (A) Restriction endonuclease

digestion of PCR fragments derived from gfp DNA and mRNA sequences. (B) Schematic

diagram of the gfp coding sequence. The positions of the restriction endonuclease

cleavage sites that were used for analysis of PCR products are indicated, and these are

numbered according to the coding region of gfp.

The shortened RT-PCR product was cloned and sequenced, and a deletion of 84 nt was

located between residues 380–463 of the GFP coding sequence.

It is likely that this 84-nt region of the jellyfish gfp cDNA sequence is efficiently

recognized as an intron when transcribed in Arabidopsis, resulting in an in-frame

deletion and the production of a defective protein product, which is predicted to be 28 aa

shorter.

Haseloff et al. (1997):

- An 84-nt cryptic intron is efficiently recognized and excised from transcripts of the

GFP coding sequence.

- The cryptic intron contains sequences similar to those required for recognition of

normal plant introns.

- They modified the codon usage of the gfp gene to mutate the intron and to restore

proper expression in Arabidopsis.

- They altered the codon usage of gfp to avoid recognition of a cryptic intron.

- They demonstrated that proper expression of the fluorescent protein is restored.

- They targeted the protein to localized compartments within the cell to obtain a gfp

gene that can be used as a bright fluorescent marker for efficient transformation and

improved regeneration of Arabidopsis plants.

Gene expression cassettes that contain the wild-type sequence (gfp), modified codon

usage (mgfp4), and additional peptide targeting sequences (mgfp4-ER). The sequences

that have altered codon usage are indicated in black, and targeting sequences are

shown in dark grey

Haseloff et al. (1997) - altered codon usage of gfp

Altered codon usage in mgfp4. The cryptic intron is shown underlined with the 59 and 39

splice sites arrowed. The upper sequence corresponds to that of gfp, and the lower

nucleotide sequence is that of mgfp4. Mutated nucleotides are shown in reverse type,

and restriction endonuclease sites are shadowed.


Modification of gfp Codon Usage Haseloff et al. (1997):

1. To destroy this cryptic intron they have mutated the sequences involved in splice-site

and branchpoint recognition and decreased the AU content of the putative intron.

2. All modifications affected only codon usage, and this modified gene, mgfp4, encodes

a protein product that is identical to that of the jellyfish.

3. The mgfp4 sequence was inserted behind the 35S promoter in pBI121, and

introduced into Arabidopsis using the root transformation technique

4. It has been shown that alteration of gfp codon usage (which also alters the putative

cryptic intron sequence) leads to 20-fold increased expression in maize protoplasts

(1996)

5. There have been reports of improved GFP expression in mammalian cells after

alteration of gene codon usage (1996)

Sequences flanking the GFP coding region. Both gfp and mgfp4 are flanked by

restriction endonuclease sites for BamHI and SacI, a ribosome binding site (RBS) for

bacterial expression, and the sequence AACA upstream of the start codon for improved

plant translation. The mgfp4-ER gene cassette contains additional sequences shown in

boldface type, which comprise a 59 terminal signal peptide and 39 HDEL sequence. An

EcoRI site was used to link the signal peptide and coding sequences.


They have fused several targeting peptides to GFP and expressed these variants in

transgenic Arabidopsis plants. One, targeted to the ER, showed a substantial

improvement over the unmodified GFP. The targeted form of GFP contains an N-

terminal signal peptide derived from an Arabidopsis vacuolar basic chitinase and the C-

terminal amino acid sequence HDEL to ensure secretion and retention of the protein

within the lumen of the ER.

GAL4-GFP enhancer trap system

A system for GAL4 targeted gene expression. An enhancer trap vector bearing a

modified GAL4-VP16 gene was inserted randomly into the Arabidopsis genome by

Agrobacterium mediated transformation. Cell specific activation of the GAL4-VP16 gene

by cellular enhancer results in activation of a linked GFP gene, allowing simple

characterisation of expression patterns. Targeted expression of another gene (X) can be

induced by genetic crossing. For example, we can use the system to trigger cell ablation

by targeted expression of a toxin.

Targeted gene expression, which is based on a method widely used in Drosophila

(Brand and Perrimon, Development 118:401-415, 1993). They have used an "enhancer-

trap" strategy to generate many transgenic plants which express different patterns of a

yeast transcription activator, GAL4. A chosen target gene can then be placed under the

control of GAL4 upstream activation sequences (UAS), transformed into plants, and

maintained silently in the absence of GAL4. Genetic crosses between this single line

and any of the library of GAL4-containing lines specifically activates the target gene in a

particular tissue or cell type. http://www.haseloff-lab.org/index.html

GAL4-GFP enhancer trap system 1. A gene for a foreign transcription activator, mGAL4-VP16 gene, is inserted into the Arabidopsis genome via Agrobacterium mediated transformation. 2. The proximity of cellular enhancer elements can drive expression of mGAL4-VP16 in distinct temporal and spatial patterns in the plant. 3. The GAL4-VP16 protein will activate transcription from a promoter containing GAL4 upstream activator sequences (UAS). A linked GAL4-responsive mgfp5-ER gene (green) marks the GAL4-expressing cells with green fluorescence. This enables primary transformants to be directly screened for patterns of GAL4 expression. 4. Any chosen gene(s) can then be crossed with a GAL4-GFP expressing line, and specifically transactivated in the GAL4-expressing cells.

http://www.haseloff-lab.org/index.html

- They found that GAL4 is not expressed in Arabidopsis due to a high A/T content,

which can interfere with mRNA processing in plants.

- They have altered the codon usage of a GAL4-VP16, so that it is expressed efficiently

in plants, and randomly inserted the modified gene into the Arabidopsis genome,

using Agrobacterium tumefaciens-mediated transformation.

- The transformation vector was designed so that expression of the GAL4-VP16 gene

would be dependent upon the fortuitous proximity of an Arabidopsis enhancer

element.

- The inserted DNA also contained a GAL4-responsive green fluorescent protein (GFP)

gene. Thus, interesting "enhancer-trap" patterns of GAL4 gene expression were

immediately and directly visible, with each GAL4-expressing cell marked by bright

green fluorescence.

They created a collection of 250 Arabidopsis lines with distinct and stable patterns of

GAL4-VP16 and GFP expression in the root. These lines provide a valuable set of

markers, where particular cell types are tagged and can be visualised with

unprecedented ease and clarity in living plants.

GAL4-VP16 expression within these lines can be used for targeted gene expression

experiments. They have produced transgenic plants which maintain regulatory proteins

or toxins, silent behind a GAL4-responsive promoter. They can now activate these

genes in specific cells by crossing to a chosen GAL4-VP16 expressing line.


Systems for targeted gene expression in plants.

Library of transgenic Arabidopsis lines which stably express artificial transcription

regulators, based on the GAL4 and HAP1 proteins from yeast. The expression of these

regulators is targeted to particular cell types - and can be used to engineer gene

expression in those cells. These enhancer trap lines show coordinate expression of a

linked GFP or CFP gene.

Selected Arabidopsis GAL4-VP16 enhancer trap lines.




Arabidopsis enhancer trap lines were previously generated through transformation with a T-DNA vector containing

a modified GAL4-VP16 transcription activator gene and a GAL4-responsive mGFP5 gene under the control of

GAL4 UASs (Haseloff, 1999). Expression of the GAL4-VP16 gene is dependent on the proximity of Arabidopsis

genomic enhancer elements, and the GAL4 protein then binds to UAS to drive tissue-specific expression of

mGFP5


Wenzel et al., Journal of Experimental Botany, Vol. 63, No. 14, pp. 5351–5364, 2012

Higaki et al., 2012 SCIENTIFIC REPORTS | 2 : 405 | DOI: 10.1038/srep00405

Higaki et al., 2012 SCIENTIFIC REPORTS | 2 : 405 | DOI: 10.1038/srep00405

http://podb.nibb.ac.jp/Organellome/

Mano et al., 2011. The Plant Organelles Database 2 (PODB2): An Updated Resource Containing Movie Data of

Plant Organelle Dynamics. Plant Cell Physiol. 52: 244–253

https://gfp.dpb.carnegiescience.edu/index.html

https://www.origene.com/products/cdna-clones/organelle-marker

Rekombinantní GFP technologie a jejich využitícr-hana.upol.cz/cellbiol/bilder/GFPT/GFPT5.pdfefficient translation of the transcribed gene in E. coli 5. The sequence AACA was inserted

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