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Molecular cloning and sequencing of a cDNA for olfactory marker protein
Abstract:
cDNA clones corresponding to mRNA for rat olfactory marker protein (OMP) were isolated from a cDNA library. The library was constructed from olfactory mucosa poly(A)+ RNA enriched for OMP mRNA and cloned into a pBR322-derived plasmid, pMG5. OMP cDNA clones were detected by using a 17-base oligonucleotide probe that contained all 16 possible sequences coding for a known partial amino acid sequence of rat OMP. The identity of these clones was confirmed by hybrid-selected translation and nucleotide sequencing. The sequence of one clone was determined and contained the complete OMP coding region of 486 nucleotides followed by 1630 nucleotides of the 3' untranslated region. The 3' untranslated region included the polyadenylylation signal 16 nucleotides upstream of the poly(A) tail. No other ATG-initiated open reading frame larger than 20 codons was present in register. RNA blot analysis of olfactory mucosa poly(A)+ RNA using this clone as a probe indicated that the level of OMP mRNA, but not its size, declined significantly within a few days following olfactory bulbectomy. OMP mRNA was not detected in 14 nonolfactory rat tissues. Surprisingly, a small amount of OMP mRNA was observed in olfactory bulb. The presence of OMP mRNA in olfactory bulb was confirmed by in vitro translation and immunoprecipitation. These results suggest either that a previously undescribed population of neurons in the olfactory bulb synthesize OMP or that OMP mRNA is transported to the bulb by axonal transport.
Insights
Researchers isolated rat olfactory marker protein (OMP) cDNA clones to study OMP mRNA regulation. OMP mRNA levels decrease after olfactory bulb removal, with unexpected OMP mRNA found in the olfactory bulb itself.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- Olfactory marker protein (OMP) is a key protein in olfactory neurons.
- Understanding OMP gene expression is crucial for olfactory system research.
Purpose of the Study:
- To isolate and characterize cDNA clones for rat OMP.
- To investigate the regulation of OMP mRNA levels and its presence in different tissues.
Main Methods:
- Construction and screening of a rat olfactory mucosa cDNA library.
- Oligonucleotide probing, hybrid-selected translation, and nucleotide sequencing.
- RNA blot analysis, olfactory bulbectomy, and in vitro translation/immunoprecipitation.
Main Results:
- Isolation of complete OMP coding region and 3' untranslated region cDNA clones.
- Significant decline in OMP mRNA levels in olfactory mucosa post-bulbectomy.
- Detection of OMP mRNA in olfactory bulb, a non-olfactory mucosa tissue.
Conclusions:
- OMP mRNA regulation is dependent on the olfactory bulb.
- The presence of OMP mRNA in the olfactory bulb suggests novel neuronal populations or axonal transport mechanisms.