A novel interaction between the 5′‐untranslated region of eukaryotic messenger RNAs and non‐contiguous sequences in the 18 S ribosomal RNA is proposed. The small ribosomal RNA contains, at its 3′‐terminus, a heavily conserved hairpin structure. It is suggested that mRNA 5′‐leader sequence stabilises this structure by interacting with other conserved nucleotides which flank it. Sequences closely related to the required sequence (A—U—C—C—A—C—C) occur quite commonly in eukaryotic mRNAs and are often found immediately upstream from the AUG‐codon. This interaction may have a role in the events which lead up to the initiation of protein synthesis.
The mRNA start site of a cloned rainbow trout protamine gene (TPG-3) has been localised using S1-nuclease mapping and primer extension of in vivo synthesised trout testis poly A+-RNA. The presumptive cap site occurs within an AT-rich region, only 14 nucleotides from the start of the protein-coding sequence. Transcription of this protamine gene in vitro, using the Hela whole-cell extract system, generates products initiated at the same nucleotide as that used in vivo. In vitro transcription is abolished by deletion of sequences between -20 and -48, within which is a canonical TATA-box having an llbp homology with the strong chick conalbumin and Adenovirus-2 major late promoters (CTATAAAAGGG).
The compound 2-bromo-4-methylphenol was identified as being responsible
for a “chemical (phenolic)”
taint in brine-salted Gouda cheese manufactured over a 3 month period.
The concentration on the
surface of cheese manufactured at the beginning of the period was
determined to be between 14
and 25 μg/kg (ppb) decreasing to between 1 and 2 μg/kg at the end.
It was proposed that
4-methylphenol and active bromine could be the precursors.
Attention focused on the brine, which
was shown to be a source of 4-methylphenol and a potential source of
bromine. Irradiation of the
brine with UV light and dosing with sodium hypochlorite solution were
considered to be important
factors in generating active bromine. Experiments, carried out in
model systems, demonstrated
that 2-bromo-4-methylphenol could be generated from sodium bromide and
4-methylphenol in the
presence of either UV light or sodium hypochlorite, but not in their
absence.
Keywords: Chemical taint; Gouda cheese; brine salting taint;
2-bromo-4-methylphenol
We have compared the strength of a trout protamine gene promoter with that of the mouse beta major-globin gene by analysing the relative levels of run-off transcripts produced in a single mammalian in vitro transcription reaction. When the promoters are introduced on separate recombinant plasmids, the protamine transcripts are synthesised with much greater efficiency than those originating from the globin cap site. This enhanced transcription of the protamine gene is again observed when the promoters are applied as separate DNA fragments derived from the same recombinant plasmid. However, when the promoters are linked on a DNA fragment that includes 7 kb of DNA separating the initiation sites, then there is a marked reduction in the protamine signal relative to the globin. Deletion of a region of this fragment that contains the sequences flanking the globin gene at positions -335 to -1400 restores the enhanced protamine gene expression to the levels observed when the promoters are carried on separate DNA fragments.
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