The nucleic acid data:
IRESite Id: 456 Version: 1
Originaly submitted by: Tomáš Mašek
Reviewed by: Martin Mokrejš Last change: 2008-09-12 14:51:30
IRESite record type:
  negative_control_plasmid_with_promoter_and_without_putative_IRES
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 which comprises luciferase and Gal4 genes as the first and
the second cistron, respectively. 13G:C hair pin loop was inserted between the two cistrons to prevent
ribosome read-through.
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
The name of the promoter used to express this mRNA:
  TPI
Description of the plasmid (facultative for promoter-less plasmid records):
pFGAL4h represents yeast multi-copy expression shuttle vector and contains strong constitutive TPI promoter.
The in vivo produced transcripts are heterogeneous (due to any of promoter?/splicing?/cleavage?/breakage?):
  yes
The in vivo produced heterogeneous transcripts occur due to alternative splicing:
  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:
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:
heterogeneous_population_of_molecules_found
Integrity (uniformity) of mRNA tested using real-time quantitative polymerase chain reaction (rtqPCR):
heterogeneous_population_of_molecules_found
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:
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 DNA sequence of the plasmid in (+) orientation annotated by its secondary structure:


GenBank formatted file with annotated plasmid sequence hyperlinked from vector image map:
pFGAL4h.jpg
Plasmid sequence verified (partially/completely) by IRESite (more details in Remarks):
  plasmid sequence confirmed by IRESite curators by restriction analysis + parts by PCR + sequencing
A promoter reported in cDNA corresponding to IRES sequence:
  not tested
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: Martin Mokrejš
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: 0
Originaly submitted by: Tomáš Mašek Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
Gal4
The description of the protein encoded in this ORF:
GAL4 is a 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:
  no
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  1734-4379
Remarks:
pFGAL4h bicistronic plasmid 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.
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
Vopalensky V., Masek T., Horvath O., Vicenova B., Mokrejs M., Pospisek M. (2008) Firefly luciferase gene contains a cryptic promoter. RNA. 14(9):1720-1729
Last change to the database: 2015-04-16 16:45:23 GMT+1