The nucleic acid data:
IRESite Id: 462 Version: 2
Originaly submitted by: Václav Vopálenský
Reviewed by: Martin Mokrejš Last change: 2008-07-08 09:17:03
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 transcript produced from monocistronic plasmid pMC-env containing full-length (except very 5'-most
Adenosine) gypsy-env 5' UTR (nt from -841 to -511 of the original sequence) mRNA cloned upstream of the
beta-galactosidase.
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:
pMC-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 nt -511 of the original sequence) and beta-galactosidase reporter gene.
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:
pMC-Env.jpg
The total number of notable open-reading frames (ORFs):
  1
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:
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):
  410-3472
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 12:56:41
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):
  82-411
How IRES boundaries were determined:
experimentally_determined
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).
The translation experiments:
Translation results:
IRESite Translation Id: 503
Version: 1 Last change: 2008-07-07 12:56:41
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:
  100.000
Name of the plasmid used as the negative control.
pbeta-actin-lacZ
The relative translation efficiency in % of the negative control:
  14.700
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:
In the absence of P2A (poliovirus protease 2A), the enzymatic activity of both pMC-Env and pbeta-actin-lacZ
reached similar levels. Upon P2A expression, a drastic decrease of beta-Gal expression from the
pbeta-actin-lacZ RNA (over fivefold decrease) was observed, whereas translation driven by the env 5' UTR was
moderately stimulated.
Translational data are derived from Fig. 7B.
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