The nucleic acid data:
IRESite Id: 468 Version: 1
Originaly submitted by: Václav Vopálenský
Reviewed by: Martin Mokrejš Last change: 2008-07-08 21:33:32
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: 
Putative spliced in vivo SV40 promoter-derived transcript produced from bicistronic plasmid pstemRL 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 with a hairpin in front of the
first cistron.
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:
pstemRhifF
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?):
  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:
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:


GenBank formatted file with annotated plasmid sequence hyperlinked from vector image map:
pstemRhifF.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):
  246-1181
ORF
ORF position:   2
Version: 0
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):
  1508-3160
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: 0
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):
  1219-1512
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: 512
Version: 1 Last change: 2008-07-08 17:55:42
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:
  183.000
Name of IRES used as the positive control:
  HIF1_alpha
Name of the plasmid used as the positive control.
pRhifF
Name of the plasmid used as the negative control.
pRF
IRESite Id of the plasmid used as positive control.
  466
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:
  4.100
The size (length) of intercistronic region in the positive control:
326
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:
As a positive control was used NIH/3T3 (ATCC CRL-1658) cell line transformed by similar vector pRhifF lacking
the stem-loop in front of the first cistron. The hairpin loop impairs translation from the first cistron while
translation from the second cistron is not affected. Thus, the relative ratio 2nd/1st citron increases.

Translational data are derived from Fig. 4A.
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: 513
Version: 1 Last change: 2008-07-08 17:55:42
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:
  296.000
Name of IRES used as the positive control:
  HIF1_alpha
Name of the plasmid used as the positive control.
pRhifF
Name of the plasmid used as the negative control.
pRF
IRESite Id of the plasmid used as positive control.
  466
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:
  1.600
The size (length) of intercistronic region in the positive control:
326
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:
As a positive control was used HeLa (ATCC CCL-2) cell line transformed by similar vector pRhifF
lacking the stem-loop in front of the first cistron. The hairpin loop impairs translation from the first
cistron while translation from the second cistron is not affected. Thus, the relative ratio 2nd/1st citron
increases.
Translational data are derived from Fig. 4A.
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: 514
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:
  113.000
Name of IRES used as the positive control:
  c-myc
Name of the plasmid used as the positive control.
phpRMF
Name of the plasmid used as the negative control.
pstemRF
IRESite Id of the plasmid used as positive control.
  355
IRESite Id of the plasmid used as negative control.
  455
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:
40
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: 515
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:
  73.000
Name of IRES used as the positive control:
  c-myc
Name of the plasmid used as the positive control.
phpRMF
Name of the plasmid used as the negative control.
pstemRF
IRESite Id of the plasmid used as positive control.
  355
IRESite Id of the plasmid used as negative control.
  455
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:
40
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
Last change to the database: 2015-04-16 16:45:23 GMT+1