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| Comparison of gfp and m-gfp5 sequences. |
| (1) The jellyfish gfp sequence is shown as the upper strand (as described for pGFP10.1 in Prasher et al. (Gene 111, 229-233, 1992), except that codon 80 contains a change from CAG to CGG - resulting in replacement of a glutamine with arginine, also noted in Chalfie et al., Science 263, 802-805, 1994) The modified gfp (m-gfp5) is positioned below it. The cryptic intron which prevents proper expression of the unmodified gene is shown arrowed. Altered sequences in the m-gfp5 are highlighted in colour, and described below: (2) N-terminal signal peptide sequence from Arabidopsis thaliana basic chitinase and C-terminal HDEL sequence for retention of GFP in the endoplasmic reticulum. ER retention allows safe accumulation of fluorescence to high levels - GFP is compartmented away from the nucleoplasm. (Haseloff. J. Siemering, K.R., Prasher, D. & Hodge, S. Proc. Natl. Acad. Sci. USA) (3) nucleotides that were altered to change the codon usage of GFP, to eliminate cryptic splicing in Arabidopsis, and perhaps other plant species. These include the codon usage changes introduced in the mgfp4 gene. (Haseloff. J. Siemering, K.R., Prasher, D. & Hodge, S. Proc. Natl. Acad. Sci. USA.) (4) V163A and S175G mutations produce improved GFP fluorescence. The mutations aid folding of the apoprotein and/or cyclisation of the chromophore (Siemering, K.R. Golbik, R., Sever, R. & Haseloff, J. Current Biology 6:1653-1663, 1996). (5) I167T mutation produces altered spectral properties (Heim, Prasher & Tsien PNAS 91, 12501-12504, 1994). In combination with the V163A and S175G, this mutation produces a protein with dual excitation peaks of approximately equal intensity at 400nm and 475nm (Siemering, K.R. Golbik, R., Sever, R. & Haseloff, J. Current Biology 6:1653-1663, 1996), and can be visualised well with either long wavelength UV (eg. hand-held lamp) or blue light (eg. argon laser). |
... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...
atg aag act aat ctt ttt ctc ttt ctc atc ttt tca ctt ctc cta tca tta tcc tcg gcc m-gfp5
M K T N L F L F L I F S L L L S L S S A
Arabidopsis thaliana basic chitinase signal sequence
M
... atg agt aaa gga gaa gaa ctt ttc act gga gtt gtc cca att ctt gtt gaa tta gat gfp
gaa ttc agt aaa gga gaa gaa ctt ttc act gga gtt gtc cca att ctt gtt gaa tta gat m-gfp5
E F S K G E E L F T G V V P I L V E L D
ggt gat gtt aat ggg cac aaa ttt tct gtc agt gga gag ggt gaa ggt gat gca aca tac gfp
ggt gat gtt aat ggg cac aaa ttt tct gtc agt gga gag ggt gaa ggt gat gca aca tac m-gfp5
G D V N G H K F S V S G E G E G D A T Y
Nco I
gga aaa ctt acc ctt aaa ttt att tgc act act gga aaa cta cct gtt cca tgg cca aca gfp
gga aaa ctt acc ctt aaa ttt att tgc act act gga aaa cta cct gtt cca tgg cca aca m-gfp5
G K L T L K F I C T T G K L P V P W P T
Nde I
ctt gtc act act ttc tct tat ggt gtt caa tgc ttt tca aga tac cca gat cat atg aaa gfp
ctt gtc act act ttc tct tat ggt gtt caa tgc ttt tca aga tac cca gat cat atg aag m-gfp5
L V T T F S Y G V Q C F S R Y P D H M K
cgg cat gac ttt ttc aag agt gcc atg ccc gaa ggt tat gta cag gaa aga act ata ttt gfp
cgg cac gac ttc ttc aag agc gcc atg cct gag gga tac gtg cag gag agg acc atc ttc m-gfp5
R H D F F K S A M P E G Y V Q E R T I F
ttc aaa gat gac ggg aac tac aag aca cgt gct gaa gtc aag ttt gaa ggt gat acc ctt gfp
ttc aag gac gac ggg aac tac aag aca cgt gct gaa gtc aag ttt gag gga gac acc ctc m-gfp5
F K D D G N Y K T R A E V K F E G D T L
|
| cryptic intron
gtt aat aga atc gag tta aaa ggt att gat ttt aaa gaa gat gga aac att ctt gga cac gfp
gtc aac agg atc gag ctt aag gga atc gat ttc aag gag gac gga aac atc ctc ggc cac m-gfp5
V N R I E L K G I D F K E D G N I L G H
|
Acc I |
aaa ttg gaa tac aac tat aac tca cac aat gta tac atc atg gca gac aaa caa aag aat gfp
aag ttg gaa tac aac tac aac tcc cac aac gta tac atc atg gcc gac aag caa aag aac m-gfp5
K L E Y N Y N S H N V Y I M A D K Q K N
V I S
gga atc aaa gtt aac ttc aaa att aga cac aac att gaa gat gga agc gtt caa cta gca gfp
ggc atc aaa gcc aac ttc aag acc cgc cac aac atc gaa gac ggc ggc gtg caa ctc gct m-gfp5
G I K A N F K T R H N I E D G G V Q L A
gac cat tat caa caa aat act cca att ggc gat ggc cct gtc ctt tta cca gac aac cat gfp
gat cat tat caa caa aat act cca att ggc gat ggc cct gtc ctt tta cca gac aac cat m-gfp5
D H Y Q Q N T P I G D G P V L L P D N H
tac ctg tcc aca caa tct gcc ctt tcg aaa gat ccc aac gaa aag aga gac cac atg gtc gfp
tac ctg tcc aca caa tct gcc ctt tcg aaa gat ccc aac gaa aag aga gac cac atg gtc m-gfp5
Y L S T Q S A L S K D P N E K R D H M V
*
ctt ctt gag ttt gta aca gct gct ggg att aca cat ggc atg gat gaa cta tac aaa taa gfp
ctt ctt gag ttt gta aca gct gct ggg att aca cat ggc atg gat gaa cta tac aaa cac m-gfp5
L L E F V T A A G I T H G M D E L Y K H
... ... ... ...
gac gaa ctc taa m-gfp5
D E L *
|