The nucleic acid data:
IRESite Id: 240 Version: 0
Originaly submitted by: Martin Mokrejš
Reviewed by: Martin Mokrejš
IRESite record type:
  plasmid_with_promoter_and_putative_IRES_translationally_characterized
The shape of the nucleic acid molecule translated:
  linear
The quality of the mRNA/+RNA sequence:
  3UTR_possibly_incomplete
The mRNA/+RNA description: 
Putative bicistronic mRNA molecule coding for eBFP and eGFP with the intercistronic region containing ELH
IRES.
The mRNA/+RNA sequence represented in the +DNA notation:


Credibility of mRNA sequence:
  reverse_engineered_sequence_and_should_match_experiment_except_3UTR
The name of the plasmid:
pNEX3_based-eBFP-ELH-eGFP
The name of the promoter used to express this mRNA:
  RSV
The in vivo produced transcripts are heterogeneous (due to any of promoter?/splicing?/cleavage?/breakage?):
  no
The in vivo produced heterogeneous transcripts occur due to alternative splicing:
  no
A promoter reported in cDNA corresponding to IRES sequence:
  no
Summary of possible issues when IRES cDNA is experimentally transcribed in vivo:
Summary of experiments studying integrity of the in vivo transcripts in a particular host:
Integrity (uniformity) of mRNA tested using Northern-blot:
not_tested
Integrity (uniformity) of mRNA tested using RNase protection:
not_tested
Integrity (uniformity) of mRNA tested using 5'-RACE:
not_tested
Integrity (uniformity) of mRNA tested using primer extension :
not_tested
Integrity (uniformity) of mRNA tested using RT-PCR:
not_tested
Integrity (uniformity) of mRNA tested using real-time quantitative polymerase chain reaction (rtqPCR):
not_tested
Integrity (uniformity) of mRNA tested using RNAi:
not_tested
Integrity (uniformity) of mRNA tested using S1 nuclease mapping:
not_tested
Cryptic promoter presence was confirmed by expression from a promoter-less plasmid:
no_promoter_confirmed
Cryptic promoter presence was confirmed in an experimental setup involving inducible promoter:
not_tested
Integrity (uniformity) of mRNA molecules or possible promoter presence expressed in vivo was tested using another method, please specify in Remarks:
not_tested
The organism used:
Aplysia californica
The abbreviated name of the donor gene or virus from which this IRES was excised and inserted into the plasmid:
ELH
The origin of IRES in the plasmid:
  nuclear
The donor organism of the IRES segment:
Aplysia californica
The DNA sequence of the plasmid in (+) orientation annotated by its secondary structure:


GenBank formatted file with annotated plasmid sequence hyperlinked from vector image map: pNEX3_based-eBFP-ELH-eGFP.jpg
The total number of notable open-reading frames (ORFs):
  2
Notable Open-Reading Frames (ORFs; protein coding regions) in the mRNA/+RNA sequence:
ORF
ORF position:   1
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
eBFP
The description of the protein encoded in this ORF:
Enhanced blue fluorescent protein
The translational frameshift (ribosome slippage) involved:
  0
The ribosome read-through involved:
  no
The alternative forms of this protein occur by the alternative initiation of translation:
  not tested
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  71-790
ORF
ORF position:   2
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
eGFP
The description of the protein encoded in this ORF:
Enhanced green fluorescent protein
The translational frameshift (ribosome slippage) involved:
  0
The ribosome read-through involved:
  no
The alternative forms of this protein occur by the alternative initiation of translation:
  not tested
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  1162-1881
Remarks:
The transcription initiation site has been annotated based on the knowledge from GenBank records AF052428 and
X77629.
Citations:
Dyer J. R., Michel S., Lee W., Castellucci V. F., Wayne N. L., Sossin W. S. (2003) An activity-dependent switch to cap-independent translation triggered by eIF4E dephosphorylation. Nat. Neurosci. 6(3):219-220
IRESs:
IRES:
Version: 2 Last change: 2007-01-23 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  ELH
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  842-1160
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  52
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  370
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -320
3'-end of IRES relative to first base of the START codon of the downstream ORF:
  -2
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
Although the article states the 5'-UTR of ELH (bases 1-313) have been cloned into the vector W. Sossin who
provided us with the sequence annotated 319nt as the ELH 5'-UTR insert. We preferred the later annotation.
Citations:
Dyer J. R., Michel S., Lee W., Castellucci V. F., Wayne N. L., Sossin W. S. (2003) An activity-dependent switch to cap-independent translation triggered by eIF4E dephosphorylation. Nat. Neurosci. 6(3):219-220
The translation experiments:
Translation results:
IRESite Translation Id: 298
Version: 2 Last change: 2007-05-15 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The translation method used to study IRES function:
in vivo
The organism used for translation:
Aplysia californica
The temperature (in degrees of Celsia):
20
The relative translation efficiency in % of this IRES:
  100.000
Name of the plasmid used as the negative control.
LR
The relative translation efficiency in % of the negative control:
  17.800
The effect of 5'-cap analogs on translation:
not tested
Rapamycin affects translation:
not tested
Type of RNA subject to translation:
  endogenous_nuclear_RNA_Pol_II_transcript
Remarks:
The exact experimental temperature has not been stated in the article. However, it seems A. californica
lays eggs in the temperature around 20 oC, while about 15 oC does not. The egg-laying activity switch is
attributed to the presence of the egg-laying hormone. The sequence of the negative control plasmid with
reverse-oriented ELH insert is not available. Data from Suppl. Fig. 1.
Citations:
Dyer J. R., Michel S., Lee W., Castellucci V. F., Wayne N. L., Sossin W. S. (2003) An activity-dependent switch to cap-independent translation triggered by eIF4E dephosphorylation. Nat. Neurosci. 6(3):219-220
Last change to the database: 2011-04-08 22:43:22 GMT+1