The nucleic acid data:
IRESite Id: 461 Version: 1
Originaly submitted by: Václav Vopálenský
Reviewed by: Martin Mokrejš Last change: 2008-07-08 09:16:48
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: 
In vivo CMV driven transcript produced from bicistronic plasmid pDC-env which comprises neomycin
phosphotransferase and beta-galactosidase as the first and the second cistron respectively and full-length
(except very 5'-most Adenosine) gypsy-env 5' UTR (nt from -841 to -511 of the original sequence) mRNA inserted
in between reporters.
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:
pDC-Env
The name of the promoter used to express this mRNA:
  CMV
Description of the plasmid (facultative for promoter-less plasmid records):
Plasmid containing gypsy-env IRES (nt from -841 to -511) inserted between neomycin phosphotransferase and beta-galactosidase reporter genes.
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:
  no (not convincing)
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:
homogeneous_population_of_molecules_confirmed
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:
not_tested
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:
Homo sapiens 293T (ATCC CRL-1573)
The abbreviated name of the donor gene or virus from which this IRES was excised and inserted into the plasmid:
gypsy
The origin of IRES in the plasmid:
  nuclear
The donor organism of the IRES segment:
Drosophila melanogaster
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:
pDC-Env.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: Václav Vopálenský Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
aph
The description of the protein encoded in this ORF:
aminoglycoside-3'-O-phosphotransferase
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):
  115-909
ORF
ORF position:   2
Version: 0
Originaly submitted by: Václav Vopálenský Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
LacZ
The description of the protein encoded in this ORF:
beta-galactosidase
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):
  1434-4496
Citations:
Ronfort C., De Breyne S., Sandrin V., Darlix J. L., Ohlmann T. (2004) Characterization of two distinct RNA domains that regulate translation of the Drosophila gypsy retroelement. RNA. 10(3):504-515
IRESs:
IRES:
Version: 1 Last change: 2008-07-07 12:55:15
Originaly submitted by: Václav Vopálenský Reviewed by: Martin Mokrejš
The IRES name:
  gypsy_env
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  1106-1435
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  197
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  526
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -328
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:
Gypsy-env IRES represents the full-length (except very 5'-most Adenosine) gypsy-env 5' UTR (-330 to +1 of the
original sequence).
Citations:
Ronfort C., De Breyne S., Sandrin V., Darlix J. L., Ohlmann T. (2004) Characterization of two distinct RNA domains that regulate translation of the Drosophila gypsy retroelement. RNA. 10(3):504-515
The translation experiments:
Translation results:
IRESite Translation Id: 502
Version: 1 Last change: 2008-07-07 10:42:13
Originaly submitted by: Václav Vopálenský Reviewed by: Martin Mokrejš
The translation method used to study IRES function:
in vivo
The organism used for translation:
Homo sapiens 293T (ATCC CRL-1573)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  108.000
Name of IRES used as the positive control:
  EMCV-R
Name of the plasmid used as the positive control.
pEMCV-D260-837
IRESite Id of the plasmid used as positive control.
  460
The relative translation efficiency in % of the positive control:
  100.000
The size (length) of intercistronic region in the positive control:
769
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:
pDC-Env exhibited enhancement of the beta-Gal/neo ratio in the presence of protease P2A, indicating that the
two cistrons were not equally affected by expression of the poliovirus protease and that the RNA segment
1–330 was able to direct cap-independent translation initiation in a bicistronic context (translational data
are derived from Fig. 9A).
Translation of the pDC-Env RNA is not affected by the FMDV-L protease (see Fig. 6)
Citations:
Ronfort C., De Breyne S., Sandrin V., Darlix J. L., Ohlmann T. (2004) Characterization of two distinct RNA domains that regulate translation of the Drosophila gypsy retroelement. RNA. 10(3):504-515
Last change to the database: 2015-04-16 16:45:23 GMT+1