The nucleic acid data:
IRESite Id: 271 Version: 7
Originaly submitted by: Martin Mokrejš
Reviewed by: Martin Mokrejš Last change: 2008-06-04 22:03:54
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:
  our_best_guess
The mRNA/+RNA description: 
Putative in vivo bicistronic T7 transcript with CAT and GFP cistrons and 5'-UTR containing EMCV-R IRES
terminated by Tphi transcription terminator. The EMCV-R IRES is located between nucleotides 57-608 of the
putative mRNA and lacks the very rightmost 6 bases immediately preceding the initiator AUG codon of EMCV
polyprotein. A putative ERAV IRES is located in the intercistronic region and although it is referred
to as 1-961 it consists of only 956 bp.
The mRNA/+RNA sequence represented in the +DNA notation:


Credibility of mRNA sequence:
  end-to-end_sequence_reverse_engineered_and_should_match_experiment
The name of the plasmid:
pE1(1-961)
The name of the promoter used to express this mRNA:
  T7
The in vivo produced transcripts are heterogeneous (due to any of promoter?/splicing?/cleavage?/breakage?):
  not tested
The in vivo produced heterogeneous transcripts occur due to alternative splicing:
  not tested
A promoter reported in cDNA corresponding to IRES sequence:
  not tested
The abbreviated name of the donor gene or virus from which this IRES was excised and inserted into the plasmid:
ERAV
The origin of IRES in the plasmid:
  viral
The donor organism of the IRES segment:
Equine rhinitis A virus 1 393/76
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:
pE1(1-961).jpg
The total number of notable open-reading frames (ORFs):
  6
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:
CAT
The description of the protein encoded in this ORF:
chloramphenicol acetyltransferase
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):
  615-1274
ORF
ORF position:   2
Version: 4 Last change: 2007-04-11 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
Lab-GFP
The description of the protein encoded in this ORF:
green fluorescent protein artificially extended at its N-terminus by some 40 aminoacid residues
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:
  yes
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  2140-3063
ORF
ORF position:   3
Version: 1 Last change: 2007-04-11 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
Lab-GFP
The description of the protein encoded in this ORF:
green fluorescent protein artificially extended at its N-terminus by some 39 aminoacid residues
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:
  yes
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  2143-3063
ORF
ORF position:   4
Version: 1 Last change: 2007-04-11 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
Lb-GFP
The description of the protein encoded in this ORF:
green fluorescent protein artificially extended at its N-terminus by some 19 aminoacid residues
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:
  yes
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  2203-3063
ORF
ORF position:   5
Version: 1 Last change: 2007-04-11 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
Lb-GFP
The description of the protein encoded in this ORF:
green fluorescent protein artificially extended at its N-terminus by some 18 aminoacid residues
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:
  yes
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  2206-3063
ORF
ORF position:   6
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
GFP
The description of the protein encoded in this ORF:
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:
  yes
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  2260-3063
Remarks:
It appeared the ERAV 5'-UTR region downstream the poly(C) tract contains 5 AUG codons. The first 4 are in
pairs adjacent to each other, while the fifth is the most downstream and is alone. Three types of proteins
were detected from complete ERAV IRES 1-961, called Lab-GFP, Lb-GFP, GFP. The naming Lab and Lb reflects
the fact that possibly viral proteases Lab and Lb are initiated at those respective AUG codons.

The Lab-GFP, Lb-GFP, and GFP ORFs are located in the putative mRNA sequence at positions:
Lab-GFP: 2143-3063 [11.8% of first cistron, i.e. CAT]
         2140-3063 [1.6% of CAT])
Lb-GFP: 2206-3063
        2203-3063 [1.4% both together]
GFP: 2260-3063 [6.7% of CAT]

These data were obtained for pE1(245-961) plasmid transcripts but should be applicable to pE1(1-961) as well
(Fig. 5B).
Citations:
Hinton T. M., Li F., Crabb B. S. (2000) Internal ribosomal entry site-mediated translation initiation in equine rhinitis A virus: similarities to and differences from that of foot-and-mouth disease virus. J. Virol. 74(24):11708-11716
IRESs:
IRES:
Version: 2 Last change: 2007-04-10 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  ERAV_1-961
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  1291-2246
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  17
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  972
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -849
3'-end of IRES relative to first base of the START codon of the downstream ORF:
  106
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
Part of the ERAV 5'-UTR region immediately downstream the poly(C) tract and counted downstream from the
poly(C) region.
Citations:
Hinton T. M., Li F., Crabb B. S. (2000) Internal ribosomal entry site-mediated translation initiation in equine rhinitis A virus: similarities to and differences from that of foot-and-mouth disease virus. J. Virol. 74(24):11708-11716
The translation experiments:
Translation results:
IRESite Translation Id: 349
Version: 1 Last change: 2007-03-29 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:
Homo sapiens BHK-21 (ATCC CCL-10)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  100.000
Name of the plasmid used as the negative control.
pT7CG
IRESite Id of the plasmid used as negative control.
  270
The relative translation efficiency in % of the negative control:
  21.000
The size (length) of intercistronic region in the negative control:
22
The effect of 5'-cap analogs on translation:
not tested
Rapamycin affects translation:
not tested
Type of RNA subject to translation:
  endogenous_cytoplasmic_uncapped_T7_transcript_without_polyA_tail
Remarks:
BHK-21 cells were infected by recombinant vaccinia virus that expresses T7 polymerase (vTF7-3). Data from Fig.
3B.
Citations:
Hinton T. M., Li F., Crabb B. S. (2000) Internal ribosomal entry site-mediated translation initiation in equine rhinitis A virus: similarities to and differences from that of foot-and-mouth disease virus. J. Virol. 74(24):11708-11716
Translation results:
IRESite Translation Id: 365
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The translation method used to study IRES function:
in vitro
The in vitro translation system:
rabbit reticulocytes lysate
The organism used for translation:
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  31.000
Name of IRES used as the positive control:
  EMCV_type_R
Name of the plasmid used as the positive control.
pEMCV
IRESite Id of the plasmid used as positive control.
  292
The relative translation efficiency in % of the positive control:
  100.000
The size (length) of intercistronic region in the positive control:
567
The effect of 5'-cap analogs on translation:
not tested
Rapamycin affects translation:
not tested
Type of RNA subject to translation:
  endogenous_cytoplasmic_uncapped_T7_transcript_without_polyA_tail
Remarks:
Fig. 5E.
Citations:
Hinton T. M., Crabb B. S. (2001) The novel picornavirus Equine rhinitis B virus contains a strong type II internal ribosomal entry site which functions similarly to that of Encephalomyocarditis virus. J. Gen. Virol. 82(Pt 9):2257-2269
Translation results:
IRESite Translation Id: 366
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The translation method used to study IRES function:
in vitro
The in vitro translation system:
rabbit reticulocytes lysate
The organism used for translation:
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  45.000
Name of IRES used as the positive control:
  EMCV_type_R
Name of the plasmid used as the positive control.
pEMCV
IRESite Id of the plasmid used as positive control.
  292
The relative translation efficiency in % of the positive control:
  100.000
The size (length) of intercistronic region in the positive control:
567
The effect of 5'-cap analogs on translation:
not tested
Rapamycin affects translation:
not tested
Type of RNA subject to translation:
  endogenous_cytoplasmic_uncapped_T7_transcript_without_polyA_tail
Remarks:
Fig. 5F.
Citations:
Hinton T. M., Crabb B. S. (2001) The novel picornavirus Equine rhinitis B virus contains a strong type II internal ribosomal entry site which functions similarly to that of Encephalomyocarditis virus. J. Gen. Virol. 82(Pt 9):2257-2269
Last change to the database: 2015-04-16 16:45:23 GMT+1