The nucleic acid data:
IRESite Id: 496 Version: 1
Originaly submitted by: Martin Mokrejš
Reviewed by: Martin Mokrejš Last change: 2008-07-15 14:46:43
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:
  3UTR_possibly_incomplete
The mRNA/+RNA description: 
Putative spliced in vivo transcript of pR-deltaEMCV-F from SV40 promoter with -400 to -1 insert of putative
LINE-1 IRES with cryptic promoter (probably within -400 to -200 region).
The mRNA/+RNA sequence represented in the +DNA notation:


Warning: mRNA sequence when devoid of trailing 'A's is still not a substring of the plasmid sequence. Is it because an intron is spliced out? Stay calm then. :-)
Credibility of mRNA sequence:
  reverse_engineered_sequence_and_should_match_experiment_maybe_except_3UTR
The name of the plasmid:
pR-deltaEMCV-L1_ORF1_-400-1-F
The name of the promoter used to express this mRNA:
  SV40
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:
  yes
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:
not_tested
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:
promoter_confirmed
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:
Mus musculus
The abbreviated name of the donor gene or virus from which this IRES was excised and inserted into the plasmid:
L1spa
The origin of IRES in the plasmid:
  nuclear
The donor organism of the IRES segment:
Mus musculus
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:
pR-deltaEMCV-L1_ORF1_-400-1-F.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:
RLuc
The description of the protein encoded in this ORF:
Renilla luciferase
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):
  192-1127
ORF
ORF position:   2
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
FLuc
The description of the protein encoded in this ORF:
Firefly luciferase
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):
  2050-3702
Remarks:
The cryptic promoter is located somewhere in the region -400 to -200.
Citations:
Li P. W., Li J., Timmerman S. L., Krushel L. A., Martin S. L. (2006) The dicistronic RNA from the mouse LINE-1 retrotransposon contains an internal ribosome entry site upstream of each ORF: implications for retrotransposition. Nucleic. Acids Res. 34(3):853-864
IRESs:
IRES:
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  LINE1-ORF1-400-1
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  1648-2047
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  521
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  920
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -402
3'-end of IRES relative to first base of the START codon of the downstream ORF:
  -3
The sequence of IRES region aligned to its secondary structure (if available):


Citations:
Li P. W., Li J., Timmerman S. L., Krushel L. A., Martin S. L. (2006) The dicistronic RNA from the mouse LINE-1 retrotransposon contains an internal ribosome entry site upstream of each ORF: implications for retrotransposition. Nucleic. Acids Res. 34(3):853-864
The translation experiments:
Translation results:
IRESite Translation Id: 546
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The translation method used to study IRES function:
in vivo
The organism used for translation:
Mus musculus ltk-
The temperature (in degrees of Celsia):
47
The relative translation efficiency in % of this IRES:
  143.000
Name of IRES used as the positive control:
  CrPV_IGR_IRES
Name of the plasmid used as the positive control.
pR-deltaEMCV-CrPV-F
Name of the plasmid used as the negative control.
pR-deltaEMCV-F
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  9.000
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:
Data from Figure 6A are affected by the cryptic promoter.
Citations:
Li P. W., Li J., Timmerman S. L., Krushel L. A., Martin S. L. (2006) The dicistronic RNA from the mouse LINE-1 retrotransposon contains an internal ribosome entry site upstream of each ORF: implications for retrotransposition. Nucleic. Acids Res. 34(3):853-864
Last change to the database: 2015-04-16 16:45:23 GMT+1