The nucleic acid data:
IRESite Id: 137 Version: 3
Originaly submitted by: Martin Mokrejš
Reviewed by: Martin Mokrejš Last change: 2008-12-16 00:47:02
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:
  both_UTRs_incomplete
The mRNA/+RNA description: 
Incomplete reverse engineered bicistronic mRNA molecule coding for LacZ and Luc reporter genes, with putative
Hairless IRES segment in the intercistronic region in which the M2 START codon is replaced by STOP codon.
Thus, two protein products are produced from this plasmid. Albeit huge the sequence provided here contains
just the two CDS and the intercistronic region.
The mRNA/+RNA sequence represented in the +DNA notation:


Warning: available plasmid sequence is exactly same as the mRNA sequence. It seems we never managed to reconstruct the original plasmid sequence used to express the studied mRNA.
Credibility of mRNA sequence:
  only_fragment_published_or_from_author_and_the_rest_is_a_guess
The name of the plasmid:
IS
The name of the promoter used to express this mRNA:
  actin_5C
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:
Hairless
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:
IS.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: 1 Last change: 2007-09-04 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
LacZ-Hairless-fusion
The description of the protein encoded in this ORF:
beta-galactosidase fused to few aminoacid residues of Hairless
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):
  1-3093
ORF
ORF position:   2
Version: 1 Last change: 2007-09-04 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
Hairless-Luc-fusion
The description of the protein encoded in this ORF:
20 aminoacid residues from 3'-end of the putative Hairless IRES sequence (initiated by M3 start codon) fused with 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):
  3478-5187
Remarks:
The plasmid sequence is not complete and could have not been completed even upon communication with Dieter
Maier. The sequence available was partly reconstructed from public sequences or reporter genes and of regions
actually cloned/verified by D. Maier. The actin 5C promoter is constitutive.
Citations:
Maier D., Nagel A. C., Preiss A. (2002) Two isoforms of the Notch antagonist Hairless are produced by differential translation initiation. Proc. Natl. Acad. Sci. U. S. A. 99(24):15480-15485
IRESs:
IRES:
Version: 1 Last change: 2007-09-04 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  Hairless
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  3082-3516
How IRES boundaries were determined:
experimentally_determined
The sequence of IRES region aligned to its secondary structure (if available):


Citations:
Maier D., Nagel A. C., Preiss A. (2002) Two isoforms of the Notch antagonist Hairless are produced by differential translation initiation. Proc. Natl. Acad. Sci. U. S. A. 99(24):15480-15485
The translation experiments:
Translation results:
IRESite Translation Id: 98
Version: 5 Last change: 2007-09-07 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:
Drosophila melanogaster S2
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  74.200
Name of IRES used as the positive control:
  Antp
Name of the plasmid used as the positive control.
CI
Name of the plasmid used as the negative control.
pA_LL
IRESite Id of the plasmid used as negative control.
  136
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  0.800
The size (length) of intercistronic region in the negative control:
29
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:
The galactosidase and luciferase activities were determined after 2 days in cellular lysates. Both
galactosidase and luciferase protein activities were measured spectrophotometrically and luminometrically,
respectively (Fig. 2B left). Presence of both proteins was confirmed immunologically from Western blots (Fig.
2B right). Raw Luc measurements published in the paper:

Construct I	2 584 684  (pAC5.1B based monocistronic plasmid with constitutive actin 5C promoter producing
		2 580 534   lacZ-Hairless_IRES-luc fusion product)

Construct IS	  15 096   avg. 15 564, 74.2% (pAC5.1B based bicistronic plasmid with constitutive actin 5C
                  16 032                       promoter producing IRES-Luc fusion product)

Construct CI      19 733   avg. 20978, 100% (SV40 promoter based plasmid with CAT-Antp_IRES-Luc, not in
alias pBGLPLCys   22 223                     IRESite)

Construct BT I     1 573   avg. 1 528, 7.2% (pBluescript based promoter-less plasmid (with/without bacterial
 		   1 483                     Lac promoter?), not in IRESite)

Construct pA LL      158   avg. 163.5, 0.8% (empty pBluescript-based monocistronic (Lac promoter) plasmid with
                     169                     very short intercistronic sequence with LacZ-luc ORFs although
Luc                                              should not be translated)
Citations:
Maier D., Nagel A. C., Preiss A. (2002) Two isoforms of the Notch antagonist Hairless are produced by differential translation initiation. Proc. Natl. Acad. Sci. U. S. A. 99(24):15480-15485
Last change to the database: 2015-04-16 16:45:23 GMT+1