The nucleic acid data:
IRESite Id: 466 Version: 2
Originaly submitted by: Václav Vopálenský
Reviewed by: Martin Mokrejš Last change: 2008-07-08 17:59:28
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:
  hopefully_full-length_mRNA
The mRNA/+RNA description: 
Putative spliced in vivo SV40 promoter-derived transcript produced from bicistronic plasmid pRL which
comprises renilla and firefly luciferases as the first and the second cistron, respectively and the part of
mouse hif-1alpha 5' UTR (nt from -289 to nt +5 of the original sequence) mRNA.
The sequence ends at its 3'-end right after the poly(A) signal from SV40 mRNA and thus the 3'-UTR might be
slightly wrong.
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:
  end-to-end_sequence_reverse_engineered_and_should_match_experiment
The name of the plasmid:
pRhifF
The name of the promoter used to express this mRNA:
  SV40
Description of the plasmid (facultative for promoter-less plasmid records):
Plasmid containing part of mouse hif-1aplha 5' UTR (nt from -289 to nt +5 of the original sequence) inserted between renilla and firefly luciferase reporter genes.
The in vivo produced transcripts are heterogeneous (due to any of promoter?/splicing?/cleavage?/breakage?):
  no (not convincing)
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:
heterogeneous_population_of_molecules_found
Integrity (uniformity) of mRNA tested using RNase protection:
homogeneous_population_of_molecules_confirmed
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 NIH/3T3 (ATCC CRL-1658)
The abbreviated name of the donor gene or virus from which this IRES was excised and inserted into the plasmid:
HIF1_alpha
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:


Plasmid sequence verified (partially/completely) by IRESite (more details in Remarks):
  plasmid sequence confirmed by IRESite curators by restriction analysis + parts by PCR + sequencing
GenBank formatted file with annotated plasmid sequence hyperlinked from vector image map:
pRhifF.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:
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: 1 Last change: 2008-07-08 16:48:36
Originaly submitted by: Václav Vopálenský Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
FFLuc
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):
  1454-3106
Remarks:
IRESite notes about verification of plasmid sequence:

The complete sequence of IRES element was determined by sequencing using Fluc_seq_reverse
(AGGAACCAGGGCGTATCTC) primer.
Citations:
Lang K. J. D., Kappel A., Goodall G. J. (2002) Hypoxia-inducible factor-1alpha mRNA contains an internal ribosome entry site that allows efficient translation during normoxia and hypoxia. Mol. Biol. Cell. 13(5):1792-1801
IRESs:
IRES:
Version: 1 Last change: 2008-07-08 16:48:36
Originaly submitted by: Václav Vopálenský Reviewed by: Martin Mokrejš
The IRES name:
  HIF1_alpha
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  1165-1458
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  38
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  331
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -289
3'-end of IRES relative to first base of the START codon of the downstream ORF:
  4
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
HIF1_alpha IRES represents part of the mouse hif-1alpha 5' UTR (nt from -289 to nt -1) plus first 5 nts from
coding region.
The translation experiments:
Translation results:
IRESite Translation Id: 506
Version: 0
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 HeLa (ATCC CCL-2)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  110.000
Name of IRES used as the positive control:
  c-myc
Name of the plasmid used as the positive control.
pRMF
Name of the plasmid used as the negative control.
pRF
IRESite Id of the plasmid used as positive control.
  25
IRESite Id of the plasmid used as negative control.
  184
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  0
The size (length) of intercistronic region in the positive control:
445
The size (length) of intercistronic region in the negative control:
67
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:
Translational data are derived from Fig. 5B.
Citations:
Lang K. J. D., Kappel A., Goodall G. J. (2002) Hypoxia-inducible factor-1alpha mRNA contains an internal ribosome entry site that allows efficient translation during normoxia and hypoxia. Mol. Biol. Cell. 13(5):1792-1801
Translation results:
IRESite Translation Id: 507
Version: 0
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 NIH/3T3 (ATCC CRL-1658)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  122.000
Name of IRES used as the positive control:
  c-myc
Name of the plasmid used as the positive control.
pRMF
Name of the plasmid used as the negative control.
pRF
IRESite Id of the plasmid used as positive control.
  25
IRESite Id of the plasmid used as negative control.
  184
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  0
The size (length) of intercistronic region in the positive control:
445
The size (length) of intercistronic region in the negative control:
67
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:
Translational data are derived from Fig. 5A.
Citations:
Lang K. J. D., Kappel A., Goodall G. J. (2002) Hypoxia-inducible factor-1alpha mRNA contains an internal ribosome entry site that allows efficient translation during normoxia and hypoxia. Mol. Biol. Cell. 13(5):1792-1801
Translation results:
IRESite Translation Id: 508
Version: 0
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 HEK293 (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.
pRF
IRESite Id of the plasmid used as negative control.
  184
The relative translation efficiency in % of the negative control:
  5.200
The size (length) of intercistronic region in the negative control:
67
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:
Translational data are derived from Fig. 3.
Citations:
Lang K. J. D., Kappel A., Goodall G. J. (2002) Hypoxia-inducible factor-1alpha mRNA contains an internal ribosome entry site that allows efficient translation during normoxia and hypoxia. Mol. Biol. Cell. 13(5):1792-1801
Translation results:
IRESite Translation Id: 509
Version: 0
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 NIH/3T3 (ATCC CRL-1658)
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.
pRF
IRESite Id of the plasmid used as negative control.
  184
The relative translation efficiency in % of the negative control:
  3.300
The size (length) of intercistronic region in the negative control:
67
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:
Translational data are derived from Fig. 3.
Citations:
Lang K. J. D., Kappel A., Goodall G. J. (2002) Hypoxia-inducible factor-1alpha mRNA contains an internal ribosome entry site that allows efficient translation during normoxia and hypoxia. Mol. Biol. Cell. 13(5):1792-1801
Translation results:
IRESite Translation Id: 510
Version: 0
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 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.
pRF
IRESite Id of the plasmid used as negative control.
  184
The relative translation efficiency in % of the negative control:
  1.300
The size (length) of intercistronic region in the negative control:
67
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:
Translational data are derived from Fig. 3.
Citations:
Lang K. J. D., Kappel A., Goodall G. J. (2002) Hypoxia-inducible factor-1alpha mRNA contains an internal ribosome entry site that allows efficient translation during normoxia and hypoxia. Mol. Biol. Cell. 13(5):1792-1801
Last change to the database: 2015-04-16 16:45:23 GMT+1