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
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).