The nucleic acid data:
IRESite Id: 608 Version: 0
Originaly submitted by: Martin Mokrejš Submission date: 2009-08-25 16:04:27
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:
  both_UTRs_incomplete
The mRNA/+RNA description: 
Part of the T7-derived transcript studied in in vitro assays. The 5'-UTR could have been longer but do not
know the necessary details. The intercistronic region contains 5-UTR sequence of EMCV-R virus (bp 315-843).
The mRNA/+RNA sequence represented in the +DNA notation:


Warning: available plasmid sequence is exactly same as the mRNA sequence. It seems we never managed to reconstruct the original plasmid sequence used to express the studied mRNA.
Credibility of mRNA sequence:
  reverse_engineered_fragment_and_the_rest_is_a_guess
The name of the plasmid:
H-GFP-IRESemcv-GUS
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:
EMCV-R
The origin of IRES in the plasmid:
  viral
The donor organism of the IRES segment:
Encephalomyocarditis virus Rueckert
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:
H-GFP-IRESemcv-GUS.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:
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:
  not tested
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  1-720
ORF
ORF position:   2
Version: 1 Last change: 2009-08-25 16:06:40
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
GUS
The description of the protein encoded in this ORF:
beta-glucuronidase
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):
  1316-3127
Remarks:
The intercistronic region inherited quite a long sequence from parental plasmids (64bp). The EMCV-R fragment
was inserted downstream of this spacer using EcoRI and Nco sites just in front with the GUS ORF.
Citations:
Dorokhov YL, Skulachev MV, Ivanov PA, Zvereva SD, Tjulkina LG, Merits A, Gleba YY, Hohn T, Atabekov JG (2002) Polypurine (A)-rich sequences promote cross-kingdom conservation of internal ribosome entry. Proc. Natl. Acad. Sci. U.S.A. 8(99):5301-5306
IRESs:
IRES:
Version: 2 Last change: 2011-04-08 21:37:11
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  EMCV-R_315-845
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  785-1315
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  65
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  595
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -531
3'-end of IRES relative to first base of the START codon of the downstream ORF:
  -1
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
The GFP-IRES-GUS cassette was also cloned into yeast vector PyeDP1/8-2 and expression of reporters studied in
unnamed yeast of strain 2805 grown at 30 oC! The EMCV-R IRES was found active about 10x above some negative
control.
Citations:
Dorokhov YL, Skulachev MV, Ivanov PA, Zvereva SD, Tjulkina LG, Merits A, Gleba YY, Hohn T, Atabekov JG (2002) Polypurine (A)-rich sequences promote cross-kingdom conservation of internal ribosome entry. Proc. Natl. Acad. Sci. U.S.A. 8(99):5301-5306
The translation experiments:
Translation results:
IRESite Translation Id: 634
Version: 2 Last change: 2009-08-26 12:16:33
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 HeLa (ATCC CCL-2)
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.
H-GFP-TMV_UI-GUS
IRESite Id of the plasmid used as negative control.
  611
The relative translation efficiency in % of the negative control:
  0.860
The size (length) of intercistronic region in the negative control:
78
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:
Data from Figure 2B. The cells were infected by recombinant vaccinia virus. Also GFP-TMV_UI-GUS (TMV UI 5'-UTR
omega leader) was used as a negative control (IRESiteID:611).
Citations:
Dorokhov YL, Skulachev MV, Ivanov PA, Zvereva SD, Tjulkina LG, Merits A, Gleba YY, Hohn T, Atabekov JG (2002) Polypurine (A)-rich sequences promote cross-kingdom conservation of internal ribosome entry. Proc. Natl. Acad. Sci. U.S.A. 8(99):5301-5306
Translation results:
IRESite Translation Id: 635
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:
  279.000
Name of the plasmid used as the negative control.
NO_RNA
The relative translation efficiency in % of the negative control:
  1.000
The effect of 5'-cap analogs on translation:
not tested
Rapamycin affects translation:
not tested
Type of RNA subject to translation:
  exogenous_RNA_without_cap_without_polyA_tail
Remarks:
Data from Figure 2E.
Citations:
Dorokhov YL, Skulachev MV, Ivanov PA, Zvereva SD, Tjulkina LG, Merits A, Gleba YY, Hohn T, Atabekov JG (2002) Polypurine (A)-rich sequences promote cross-kingdom conservation of internal ribosome entry. Proc. Natl. Acad. Sci. U.S.A. 8(99):5301-5306
Translation results:
IRESite Translation Id: 636
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:
wheat germ lysate
The organism used for translation:
The temperature (in degrees of Celsia):
25
The relative translation efficiency in % of this IRES:
  100.000
Name of the plasmid used as the negative control.
NO_RNA
The relative translation efficiency in % of the negative control:
  80.000
The effect of 5'-cap analogs on translation:
not tested
Rapamycin affects translation:
not tested
Type of RNA subject to translation:
  exogenous_RNA_without_cap_without_polyA_tail
Remarks:
Data from Figure 2D.
Citations:
Dorokhov YL, Skulachev MV, Ivanov PA, Zvereva SD, Tjulkina LG, Merits A, Gleba YY, Hohn T, Atabekov JG (2002) Polypurine (A)-rich sequences promote cross-kingdom conservation of internal ribosome entry. Proc. Natl. Acad. Sci. U.S.A. 8(99):5301-5306
Last change to the database: 2015-04-16 16:45:23 GMT+1