The nucleic acid data:
IRESite Id: 135 Version: 5
Originaly submitted by: Martin Mokrejš
Reviewed by: Martin Mokrejš Last change: 2008-12-16 00:51:16
IRESite record type:
  plasmid_with_promoter_and_putative_IRES_without_translational_characterization
The shape of the nucleic acid molecule translated:
  linear
The quality of the mRNA/+RNA sequence:
  both_UTRs_incomplete
The mRNA/+RNA description: 
mRNA from construct I: lacZ and luc genes with Hairless putative IRES segment with M2 Met codon mutated to
Leu.  Albeit huge the sequence provided here contains just the one polyprotein .
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:
I
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:
  no
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:
homogeneous_population_of_molecules_confirmed
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:
no_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:
Drosophila melanogaster S2
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: I.jpg
The total number of notable open-reading frames (ORFs):
  1
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:
LacZ-Hairless_IRES-luc-fusion
The description of the protein encoded in this ORF:
beta-galactosidase protein fused with part of the Hairless 5'UTR fused to luciferase. The protein had luciferase activity at least.
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):
  1-5187
Remarks:
The construct I was based on pAC5.1B (Invitrogen) and the M2 start codon present in the putative IRES segment
was replaced by Leu codon. The construct I shows very high luciferase activity, more than two orders higher
than the IS construct, where the M2 start codon was replaced with stop codon (instead of the leucine as in the
case of construct I) and so no fusion protein was produced. It might be the IRES-like activity was observed
because of fusion Gal-Luc protein being still active in I. The IS plasmid construct was used in the
experiments published in the article instead of this plasmid I. Both vectors use strong actin 5C promoter.

The BT_I plasmid (not in IRESite) construct was used to test cryptic promoter activity in the putative IRES
segment (the M2 start codon was replaced by Leu as in monocistronic-fusion I plasmid instead of being based on
the bicistronic IS plasmid which would be more tempting).

The positive control IRES used was the Antp IRES segment in CI plasmid, which sequence is completely
unavailable so far and which had different reporter genes (SV40-CAT-Antp-Luc).

Additional comment from Dieter Maier: We found in northern blots no indication for a mRNA corresponding to
the short protein. It is true that there is luc activity in the BTI (Bluescript without promoter) construct,
but much less than with the IS construct. In addition the BTI construct showed also beta Gal activity, with
other words the luc activity was maybe a result of the fusion lacZ-luc protein.  However, never say never
again, I cannot 100% exclude a week promoter in our sequence.

The actin 5C promoter was described by Bond and Davidson (1986).
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
Bond B. J., Davidson N. (1986) The Drosophila melanogaster actin 5C gene uses two transcription initiation sites and three polyadenylation sites to express multiple mRNA species. Mol. Cell. Biol. 6(6):2080-2088
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
Last change to the database: 2011-04-08 22:43:22 GMT+1