The nucleic acid data:
IRESite Id: 114 Version: 12
Originaly submitted by: Martin Mokrejš
Reviewed by: Martin Mokrejš Last change: 2007-01-23 00:00:00
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: 
Bicistronic capped T7 transcript containing Rluc, BCL2 IRES, Fluc-fusion and synthetic poly(A) tail (short,
35b).
The mRNA/+RNA sequence represented in the +DNA notation:


Credibility of mRNA sequence:
  end-to-end_sequence_completely_same_as_in_the_experiment
The name of the plasmid:
pRL-BCL2-FL-polyA
The name of the promoter used to express this mRNA:
  T7
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:
BCL2
The origin of IRES in the plasmid:
  nuclear
The donor organism of the IRES segment:
Homo sapiens HeLa (ATCC CCL-2)
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:
pRL-BCL2-FL-polyA.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):
  12-947
ORF
ORF position:   2
Version: 2 Last change: 2006-10-30 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
FLuc-fusion
The description of the protein encoded in this ORF:
Firefly luciferase fused N-terminally with 6 extra aminoacid residues (including the initiator ATG) from BCL2 protein.
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):
  2101-3771
Remarks:
Annotation of the plasmid: 16-812: CMV immediate early enhancer/promoter; 751: CMV transcription start;
869-1005: chimeric intron; 1049-1067: T7 RNA polymerase promoter (-17 - +2); 1066: T7 promoter transcription
start (+1); 1077-2012: RLUc; 2028-3165: BCL2 5'-UTR; 3166-4836: FFluc-fusion protein ORF

Authors corrections/comments: "We did utilize GI:179366 in numbering the bases for the BCL2 5'UTR. Due to the
high GC content at the 5'-end of this leader, the longest 5'-UTR we could obtain by RT-PCR was 1138bp in
length (Fig. 1). Unfortunately, the Methods/Materials was written earlier and the 1146bp was an estimate that
was later accidentally not corrected."

Negligible promoter activity was confirmed using promoter-less plasmid (IRESite Id:125) lacking
cytomegalovirus promoter with subsequent RLuc & FLuc luciferase activity detection (Fig. 3A, bottom panel).
Fig. 3B could be interpreted as either confirming cryptic promoter or aberrant splicing (which is proved in
Fig. 3C). pRL-BCL2-FL plasmid transcript is spliced into two forms, one of which is a monocistronic FLuc
transcript, making this plasmid unsuitable for DNA transfection studies.
Citations:
Sherrill K. W., Byrd M. P., Van Eden M. E., Lloyd R. E. (2004) BCL-2 translation is mediated via internal ribosome entry during cell stress. J. Biol. Chem. 279(28):29066-29074
IRESs:
IRES:
Version: 4 Last change: 2007-01-23 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  BCL2
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  963-2100
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  16
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  1153
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -1138
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):


The translation experiments:
Translation results:
IRESite Translation Id: 93
Version: 13 Last change: 2006-06-20 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The translation method used to study IRES function:
in vitro
The in vitro translation system:
rabbit reticulocytes lysate
The organism used for translation:
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  244.000
Name of IRES used as the positive control:
  HCV_type_1b
Name of the plasmid used as the positive control.
pRL-HCV-FL-polyA
Name of the plasmid used as the negative control.
pRL-FL-polyA
IRESite Id of the plasmid used as positive control.
  112
IRESite Id of the plasmid used as negative control.
  133
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:
485
The size (length) of intercistronic region in the negative control:
28
The effect of 5'-cap analogs on translation:
not tested
Rapamycin affects translation:
not tested
Type of RNA subject to translation:
  exogenous_RNA_with_GpppG_cap_with_polyA_tail
Remarks:
These data are based on Fig. 2 in the article. The measured values are hardly interpretable. In vitro
transcripts were produced using T7 polymerase in presence of m7G cap analog at ratio of 4:1 versus rGTP and
equimolar amounts of RNA were subject to translation in RRL. RNAs had short (35nt) polyA tail. pRL-FL was
used as the negative control although pRL-revH-FL would have been better. The actual expression from the
second cistron was 11% compared to the cap-dependent expression from the first cistron. The expression from
second cistron in positive control using HCV IRES had activity of 4.5% of the first cistron. Thus, the BCL2
IRES seemed to be more active then HCV IRES (about twice while in vivo experiments shown in Fig. 4 show the
opposite). The yields from the first cistron were not equal although all transcripts were capped and 5'-UTRs
had same length. One of the possible explanations might be that different efficiencies of capping account for
this. An empty vector pRL-FL-polyA was used as the negative control. pRL-HCV-FL-polyA was used as the positive
control.
Citations:
Sherrill K. W., Byrd M. P., Van Eden M. E., Lloyd R. E. (2004) BCL-2 translation is mediated via internal ribosome entry during cell stress. J. Biol. Chem. 279(28):29066-29074
Translation results:
IRESite Translation Id: 97
Version: 3 Last change: 2006-06-20 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The translation method used to study IRES function:
in vivo
The organism used for translation:
Homo sapiens HEK 293T/17 (ATCC CRL-11268)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  6.000
Name of IRES used as the positive control:
  HCV_type_1b
Name of the plasmid used as the positive control.
pRL-HCV-FL-polyA
Name of the plasmid used as the negative control.
pRL-revHCV-FL-polyA
IRESite Id of the plasmid used as positive control.
  112
IRESite Id of the plasmid used as negative control.
  130
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  0.760
The size (length) of intercistronic region in the positive control:
485
The size (length) of intercistronic region in the negative control:
485
The effect of 5'-cap analogs on translation:
not tested
Rapamycin affects translation:
not tested
Type of RNA subject to translation:
  exogenous_RNA_with_GpppG_cap_with_polyA_tail
Remarks:
These data are based on Fig. 4A. In vitro transcripts were produced using T7 polymerase in presence of m7G cap
analog at ratio of 4:1 versus rGTP and equimolar amounts of RNA transfected into cells. RNAs had short (35nt)
polyA tail. Measured values were normalized to amount of radiolabelled mRNA transfected into cells.
Citations:
Sherrill K. W., Byrd M. P., Van Eden M. E., Lloyd R. E. (2004) BCL-2 translation is mediated via internal ribosome entry during cell stress. J. Biol. Chem. 279(28):29066-29074
Last change to the database: 2015-04-16 16:45:23 GMT+1