The nucleic acid data:
IRESite Id: 86 Version: 14
Originaly submitted by: Tomáš Mašek Submission date: 2005-12-15 00:00:00
Reviewed by: Tomáš Mašek Last change: 2008-07-04 12:33:27
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: 
mRNA produced by bicistronic plasmid pFGAL4h-HCV1 which comprises luciferase and Gal4 genes as the first and
the second cistron, respectively.The full-length 5'-UTR of HCV genomic RNA together with the first 45 nt of a
viral polyprotein gene was inserted in frame into the intercistronic region of the reporter pFGAL4h vector
just behind the 13G:C hairpin, thus making the N-terminal fusion of the first 15 amino acids residues from the
HCV polyprotein with the yeast transcriptional activator Gal4p. The integrity of bicistronic transcript was
tested by RT PCR and real time PCR setups and with the use of promoter-less palsmid approach.
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:
pFGAL4h-HCV1
The name of the promoter used to express this mRNA:
  TPI
The in vivo produced transcripts are heterogeneous (due to any of promoter?/splicing?/cleavage?/breakage?):
  no
The in vivo produced heterogeneous transcripts occur due to alternative splicing:
  no
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:
unclear_result
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:
homogeneous_population_of_molecules_confirmed
Integrity (uniformity) of mRNA tested using real-time quantitative polymerase chain reaction (rtqPCR):
homogeneous_population_of_molecules_confirmed
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:
Saccharomyces cerevisiae pJ69a
The abbreviated name of the donor gene or virus from which this IRES was excised and inserted into the plasmid:
HCV1a
The origin of IRES in the plasmid:
  viral
The donor organism of the IRES segment:
Hepatitis C virus type 1a
The DNA sequence of the plasmid in (+) orientation annotated by its secondary structure:


Plasmid sequence verified (partially/completely) by IRESite (more details in Remarks):
  plasmid sequence confirmed by IRESite curators by restriction analysis + parts by PCR + sequencing
GenBank formatted file with annotated plasmid sequence hyperlinked from vector image map:
pFGAL4h-HCV1.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: Tomáš Mašek Reviewed by: Tomáš Mašek
The abbreviated name of this ORF/gene:
Fluc
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:
  no
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  1-1653
ORF
ORF position:   2
Version: 4 Last change: 2006-03-16 00:00:00
Originaly submitted by: Tomáš Mašek Reviewed by: Tomáš Mašek
The abbreviated name of this ORF/gene:
HCV1_GAL4
The description of the protein encoded in this ORF:
The fusion protein comprises the first 15 amino acids of HCV viral polyprotein and GAL4 DNA-binding transcription factor. Gal4p is required for the activation of the GAL genes in response to galactose; repressed by Gal80p and activated by Gal3p.
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:
  yes
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  2053-4752
OPTIONAL: The function of the encoded protein
Molecular Function: transcriptional activator activity (TAS)
Biological Process: galactose metabolism (TAS) regulation of transcription, DNA-dependent (TAS)
Cellular Component: nucleus (TAS)
Remarks:
Sc_HCV_type_1a IRES was inserted into intercistronic position of pFGAL4h bicistronic plasmid that has been
designed for the use in the pFGAL4h/PJ69A reporter system. pFGAL4h vector bears genes for the firefly
luciferase and Gal4p as a first and second cistron, respectively. A stable stem-loop hairpin containing 13G:C
pairs was inserted between the two genes in order to prevent ribosome read-through from the first to the
second cistron. The yeast strain PJ69A contains besides the GAL4 and GAL80 deletions also reporter genes
coding for the yeast Ade2 and His3 proteins and for the bacterial beta-galactosidase - all of them under the
control of the Gal4-inducible Gal1 promoter. Thus pFGAL4/PJ69A represents a specialised and sensitive system,
which allows an enhancement of the measured signal by in vivo coupled transcription and enzymatic detection.
This system can be used for both measuring the ratio of the beta-gal/luc enzymatic activities and searching
for IRES activity by screening the colony growth rates on selection media lacking adenine and histidine and
containing various concentrations of the competitive inhibitor of the histidine biosynthetic pathway.

Vector was created by IRESite curator - thus whole sequence is verified by above mentioned methods.
Citations:
Masek T., Vopalensky V., Horvath O., Vortelova L., Feketova Z., Pospisek M. (2007) Hepatitis C virus internal ribosome entry site initiates protein synthesis at the authentic initiation codon in yeast. J. Gen. Virol. 88(Pt 7):1992-2002
IRESs:
IRES:
Version: 5 Last change: 2008-07-04 12:26:21
Originaly submitted by: Tomáš Mašek Reviewed by: Tomáš Mašek
The IRES name:
  HCV_type_1a
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  1712-2096
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  59
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  443
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:
  43
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
Sc_HCV_type_1a IRES represents the full-length 5'-UTR of HCV RNA (type 1a) and the first 15 codons of viral
polyprotein.
Citations:
Masek T., Vopalensky V., Horvath O., Vortelova L., Feketova Z., Pospisek M. (2007) Hepatitis C virus internal ribosome entry site initiates protein synthesis at the authentic initiation codon in yeast. J. Gen. Virol. 88(Pt 7):1992-2002
The translation experiments:
Translation results:
IRESite Translation Id: 79
Version: 7 Last change: 2008-07-04 12:26:21
Originaly submitted by: Tomáš Mašek Reviewed by: Tomáš Mašek
The translation method used to study IRES function:
in vivo
The organism used for translation:
Saccharomyces cerevisiae pJ69a
The temperature (in degrees of Celsia):
28
The relative translation efficiency in % of this IRES:
  100.000
Name of the plasmid used as the negative control.
pFGAL4-L428
IRESite Id of the plasmid used as negative control.
  126
The relative translation efficiency in % of the negative control:
  3.800
The size (length) of intercistronic region in the negative control:
432
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:
Measured activity of Sc_HCV_type_1a IRES in translation initiation was 26 times higher than the almost basal
activity of vectors with lambda phage DNA sequences which were used as a negative control because of nearly
the same length of intercistronic region. The relative translation efficiency and size of intercistronic
region of negative control have been inserted as an avarage values of all negative controls used (pFGAL4-L413,
L-428, L456, IDs 126-128).
Citations:
Masek T., Vopalensky V., Horvath O., Vortelova L., Feketova Z., Pospisek M. (2007) Hepatitis C virus internal ribosome entry site initiates protein synthesis at the authentic initiation codon in yeast. J. Gen. Virol. 88(Pt 7):1992-2002
Last change to the database: 2015-04-16 16:45:23 GMT+1