The nucleic acid data:
IRESite Id: 276 Version: 3
Originaly submitted by: Martin Mokrejš
Reviewed by: Martin Mokrejš Last change: 2008-06-04 22:01:19
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 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 is retained only to the 4th
AUG codon (nucleotides 1964-1966) which starts the GFP ORF. Thus, there is no remaining sequence from
MCS between putative ERAV IRES and GFP ORF.
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(245-921)
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(245-921).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:
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:
  no
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  615-1274
ORF
ORF position:   2
Version: 0
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 21 aminoacid residues initiated from 2nd AUG codon encoded by ERAV IRES
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):
  1901-2767
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.

Plasmid constructs lacking a MCS between the cloned ERAV IRES and GFP ORF were described in Fig. 7A.
Results shown in Fig. 7C demonstrate that not all GFP protein fusion variants could be detected in
certain truncations of putative ERAV IRES. In this particular case, no plain GFP nor Lb-GFP was detected.
Authors claim the Lab-GFP corresponds in size to the one initiated in wild-type from AUG2, although chance
to distinguish from the polypeptide initiated at AUG1 which is longer by one aminoacid residue on 12%
PAA gel is questionable. It seems sequences downstream the second pair of AUG codons are required to allow
use of AUG codons 3 and 4.
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: 1 Last change: 2007-04-04 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  ERAV_245-921
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  1290-1966
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  16
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  692
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -611
3'-end of IRES relative to first base of the START codon of the downstream ORF:
  65
The sequence of IRES region aligned to its secondary structure (if available):


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: 354
Version: 1 Last change: 2007-04-04 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:
  71.000
Name of IRES used as the positive control:
  ERAV_245-961
Name of the plasmid used as the positive control.
pE1(245-961)
Name of the plasmid used as the negative control.
pT7CG
IRESite Id of the plasmid used as positive control.
  272
IRESite Id of the plasmid used as negative control.
  270
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  21.000
The size (length) of intercistronic region in the positive control:
626
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.
7B.
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
Last change to the database: 2015-04-16 16:45:23 GMT+1