The nucleic acid data:
IRESite Id: 186 Version: 6
Originaly submitted by: Martin Mokrejš
Reviewed by: Martin Mokrejš Last change: 2007-01-24 00:00:00
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:
  3UTR_possibly_incomplete
The mRNA/+RNA description: 
Bicistronic mRNA molecule transcribed from pRL-HL plasmid from the CMV promoter. The sequence of the mRNA
starts at a putative CMV transcription start site and the chimeric intron is spliced out. The sequence ends at
its 3'-end right after the poly(A) signal from bovine growth hormone (BGH) mRNA and thus the 3'-UTR might be
slightly wrong (it is unknown what region of the BGH signal could was transcribed and where the poly(A)
tail was added).
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:
  guessed_as_the_sequence_was_never_published_by_authors_nor_described_in_sufficient_detail
The name of the plasmid:
pRL-HL
The name of the promoter used to express this mRNA:
  CMV
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:
  not tested
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:
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:
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 HuH7
The abbreviated name of the donor gene or virus from which this IRES was excised and inserted into the plasmid:
HCV_type_1b
The origin of IRES in the plasmid:
  viral
The donor organism of the IRES segment:
Hepatitis C virus type 1b
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-HL.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: 2006-07-16 00:00:00
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):
  194-1129
ORF
ORF position:   2
Version: 1 Last change: 2006-07-16 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 fusion protein with 22 aminoacid residues from HCV ORF.
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):
  1557-3275
Remarks:
Integrity of mRNA transcripts in Huh-7 derived cells transfected by the pRL-HL plasmid was studied by Northern
blot with contradictory results: in RCF-26 cell line obtained a slightly longer than 3.3 kb transcript was
observed using HCV_UTR probe whereas in RCF-1 cell line the transcript size was about 4.2 kb. The 3.3 kb size
is just about the region of both ORFs and the intercistronic region (the putative mRNA sequence used in
IRESite is 3492nt long). The stability of both reporter proteins in RCF-1 cells when treated with
cycloheximide was found to be about 6 hrs (half-life). In subsequent experiments mentioned in the article the
RCF-26 cell line was used.
Citations:
Honda M., Kaneko S., Matsushita E., Kobayashi K., Abell G. A., Lemon S. M. (2000) Cell cycle regulation of hepatitis C virus internal ribosomal entry site-directed translation. Gastroenterology. 118(1):152-162
IRESs:
IRES:
Version: 1 Last change: 2006-07-16 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  HCV_type_1b+66
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  1216-1622
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  87
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  493
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -341
3'-end of IRES relative to first base of the START codon of the downstream ORF:
  65
The sequence of IRES region aligned to its secondary structure (if available):


Citations:
Honda M., Kaneko S., Matsushita E., Kobayashi K., Abell G. A., Lemon S. M. (2000) Cell cycle regulation of hepatitis C virus internal ribosomal entry site-directed translation. Gastroenterology. 118(1):152-162
Last change to the database: 2015-04-16 16:45:23 GMT+1