IRESite_Id:578 characteristics of FeLV-Notch2_1-814 IRES studied in pSV-CAT/FeLV-Notch2-1

IRESite: The database of experimentally verified IRES structures

The nucleic acid data:

IRESite Id: `578`	Version: `2`
Originaly submitted by: Martin Mokrejš	Submission date: `2009-04-09 02:49:56`
Reviewed by: Martin Mokrejš	Last change: `2009-07-16 15:26:40`

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:
only_IRES_fragment

The mRNA/+RNA description:

FeLV-Notch2 IRES fragment 1-814.

The mRNA/+RNA sequence represented in the +DNA notation:

gcgcaagtct ttgctgagac ttgaccgccc cgggtacccg tgtacgaata aacctcttgc cgattgcatc tgactcgtgg tctcggtgtt ccgtgggcgc 
^1         ^11        ^21        ^31        ^41        ^51        ^61        ^71        ^81        ^91        

ggggcctcat cgccgaggaa gacccagttc gggggtcttt catttggggg ctcgtccggg atagagaccc ccaacccccg ggaccaccga cccaccatca 
^101       ^111       ^121       ^131       ^141       ^151       ^161       ^171       ^181       ^191       

ggaggcacat ttacgcgccc tgcagctggt ccaagaagag atccagagac ctctagcggc ggcctaccga gaaaagctcg aaaccccggt tgtgcctcac 
^201       ^211       ^221       ^231       ^241       ^251       ^261       ^271       ^281       ^291       

cccttcaaac caggagactc cgtctgggtt cggagacatc aaaccaagaa cctcgagcca cggtggaaag gaccacatat cgtcctcctg accaccccca 
^301       ^311       ^321       ^331       ^341       ^351       ^361       ^371       ^381       ^391       

cagccttaaa ggtagacgga gttgctgcct ggattcacgc ctcccatgtg aaagctgcag gaccaaccac caatcaagac ctctcggaca gccccagctc 
^401       ^411       ^421       ^431       ^441       ^451       ^461       ^471       ^481       ^491       

agacgatcca tcgagatgga aagtccaacg cacccaaaac cctctaaaga taagactctc tcgtggaact tagtgtttct gctctaaggt gtttctggaa 
^501       ^511       ^521       ^531       ^541       ^551       ^561       ^571       ^581       ^591       

attgacaacc gccagtgtgt tcaagattca gaccagtgtt tcaagaacac ggatgcagca gcagctcttc tggcctctca cgccatccag gggaccttgt 
^601       ^611       ^621       ^631       ^641       ^651       ^661       ^671       ^681       ^691       

cctaccctct tgtgtccgtc gtcagtgaat ctctgactcg aaaaggtact cagcttctct atctacttgc tgttgccgtt gtcattatcc tgtttattat 
^701       ^711       ^721       ^731       ^741       ^751       ^761       ^771       ^781       ^791       

cttgctggga gttatcatg
^801       ^811

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

The name of the plasmid:
pSV-CAT/FeLV-Notch2-1_814/LUC The name of the promoter used to express this mRNA:
SV40

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:

no (not convincing)

A promoter reported in cDNA corresponding to IRES sequence:

no (not convincing)

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:
homogeneous_population_of_molecules_confirmed

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:
heterogeneous_population_of_molecules_found

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 HeLa (ATCC CCL-2)

The abbreviated name of the donor gene or virus from which this IRES was excised and inserted into the plasmid:
FeLV-Notch2

The origin of IRES in the plasmid:

viral

The donor organism of the IRES segment:

Felis silvestris 9683

The DNA sequence of the plasmid in (+) orientation annotated by its secondary structure:

gcgcaagtct ttgctgagac ttgaccgccc cgggtacccg tgtacgaata aacctcttgc cgattgcatc tgactcgtgg tctcggtgtt ccgtgggcgc 
^1         ^11        ^21        ^31        ^41        ^51        ^61        ^71        ^81        ^91        

ggggcctcat cgccgaggaa gacccagttc gggggtcttt catttggggg ctcgtccggg atagagaccc ccaacccccg ggaccaccga cccaccatca 
^101       ^111       ^121       ^131       ^141       ^151       ^161       ^171       ^181       ^191       

ggaggcacat ttacgcgccc tgcagctggt ccaagaagag atccagagac ctctagcggc ggcctaccga gaaaagctcg aaaccccggt tgtgcctcac 
^201       ^211       ^221       ^231       ^241       ^251       ^261       ^271       ^281       ^291       

cccttcaaac caggagactc cgtctgggtt cggagacatc aaaccaagaa cctcgagcca cggtggaaag gaccacatat cgtcctcctg accaccccca 
^301       ^311       ^321       ^331       ^341       ^351       ^361       ^371       ^381       ^391       

cagccttaaa ggtagacgga gttgctgcct ggattcacgc ctcccatgtg aaagctgcag gaccaaccac caatcaagac ctctcggaca gccccagctc 
^401       ^411       ^421       ^431       ^441       ^451       ^461       ^471       ^481       ^491       

agacgatcca tcgagatgga aagtccaacg cacccaaaac cctctaaaga taagactctc tcgtggaact tagtgtttct gctctaaggt gtttctggaa 
^501       ^511       ^521       ^531       ^541       ^551       ^561       ^571       ^581       ^591       

attgacaacc gccagtgtgt tcaagattca gaccagtgtt tcaagaacac ggatgcagca gcagctcttc tggcctctca cgccatccag gggaccttgt 
^601       ^611       ^621       ^631       ^641       ^651       ^661       ^671       ^681       ^691       

cctaccctct tgtgtccgtc gtcagtgaat ctctgactcg aaaaggtact cagcttctct atctacttgc tgttgccgtt gtcattatcc tgtttattat 
^701       ^711       ^721       ^731       ^741       ^751       ^761       ^771       ^781       ^791       

cttgctggga gttatcatg
^801       ^811

GenBank formatted file with annotated plasmid sequence hyperlinked from vector image map:

The total number of notable open-reading frames (ORFs):

0

Remarks:

The integrity of the transcripts was assessed by RT-PCR (Rohn et al., 1996), S1 nuclease protection (Lauring
et al., 2000, Figure 1B), Northern blot (Lauring et al., 2000, Supplemental Figure 1). Please note we do not
have sequence of the vector not even of the ORFs flanking the putative IRES.

Citations:

Lauring A. S., Overbaugh J. (2000) Evidence that an IRES within the Notch2 coding region can direct expression of a nuclear form of the protein. Mol. Cell. 6(4):939-945

IRESs:

IRES:

Version: `0`
Originaly submitted by: Martin Mokrejš	Reviewed by: Martin Mokrejš
The IRES name: `FeLV-Notch2_1-814`	The functional status of IRES: `functional`

The IRES absolute position (the range includes START and STOP codons or their equivalents):

1-814

How IRES boundaries were determined:
experimentally_determined

The sequence of IRES region aligned to its secondary structure (if available):

gcgcaagtct ttgctgagac ttgaccgccc cgggtacccg tgtacgaata aacctcttgc cgattgcatc tgactcgtgg tctcggtgtt ccgtgggcgc 
^1         ^11        ^21        ^31        ^41        ^51        ^61        ^71        ^81        ^91        

ggggcctcat cgccgaggaa gacccagttc gggggtcttt catttggggg ctcgtccggg atagagaccc ccaacccccg ggaccaccga cccaccatca 
^101       ^111       ^121       ^131       ^141       ^151       ^161       ^171       ^181       ^191       

ggaggcacat ttacgcgccc tgcagctggt ccaagaagag atccagagac ctctagcggc ggcctaccga gaaaagctcg aaaccccggt tgtgcctcac 
^201       ^211       ^221       ^231       ^241       ^251       ^261       ^271       ^281       ^291       

cccttcaaac caggagactc cgtctgggtt cggagacatc aaaccaagaa cctcgagcca cggtggaaag gaccacatat cgtcctcctg accaccccca 
^301       ^311       ^321       ^331       ^341       ^351       ^361       ^371       ^381       ^391       

cagccttaaa ggtagacgga gttgctgcct ggattcacgc ctcccatgtg aaagctgcag gaccaaccac caatcaagac ctctcggaca gccccagctc 
^401       ^411       ^421       ^431       ^441       ^451       ^461       ^471       ^481       ^491       

agacgatcca tcgagatgga aagtccaacg cacccaaaac cctctaaaga taagactctc tcgtggaact tagtgtttct gctctaaggt gtttctggaa 
^501       ^511       ^521       ^531       ^541       ^551       ^561       ^571       ^581       ^591       

attgacaacc gccagtgtgt tcaagattca gaccagtgtt tcaagaacac ggatgcagca gcagctcttc tggcctctca cgccatccag gggaccttgt 
^601       ^611       ^621       ^631       ^641       ^651       ^661       ^671       ^681       ^691       

cctaccctct tgtgtccgtc gtcagtgaat ctctgactcg aaaaggtact cagcttctct atctacttgc tgttgccgtt gtcattatcc tgtttattat 
^701       ^711       ^721       ^731       ^741       ^751       ^761       ^771       ^781       ^791       

cttgctggga gtta
^801       ^811

Citations:

Lauring A. S., Overbaugh J. (2000) Evidence that an IRES within the Notch2 coding region can direct expression of a nuclear form of the protein. Mol. Cell. 6(4):939-945

The translation experiments:

Translation results:

IRESite Translation Id: 615

Version: `0`
Originaly submitted by: Martin Mokrejš	Reviewed by: Martin Mokrejš

The translation method used to study IRES function:
in vivo

The organism used for translation:

Homo sapiens HeLa (ATCC CCL-2)

The temperature (in degrees of Celsia):
37

The relative translation efficiency in % of this IRES:

141.000

Name of IRES used as the positive control:

PV

Name of the plasmid used as the positive control. `pSV-CAT/polio/LUC`	Name of the plasmid used as the negative control. `pSV-CAT/mutEMCV/LUC`
The relative translation efficiency in % of the positive control: `100.000`	The relative translation efficiency in % of the negative control: `13.000`

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:

Figure 3B.

Citations:

Lauring A. S., Overbaugh J. (2000) Evidence that an IRES within the Notch2 coding region can direct expression of a nuclear form of the protein. Mol. Cell. 6(4):939-945

Last change to the database: 2019-03-18 09:32:49 GMT+1