Expression of calmodulin mRNA in rat olfactory neuroepithelium.

A calmodulin (CaM) cDNA was isolated by differential hybridization screening of a lambda gt10 library prepared from rat olfactory mucosa. This cDNA fragment, containing most of the open reading frame of the rat CaMI gene, was subcloned and used to characterize steady-state expression of CaM mRNA in rat olfactory neuroepithelium and bulb. Within the bulb mitral cells are the primary neuronal population expressing CaM mRNA. The major CaM mRNA expressed in the olfactory mucosa is 1.7 kb with smaller contributions from mRNAs of 4.0 and 1.4 kb. CaM mRNA was primarily associated with the olfactory neurons and, despite the cellular complexity of the tissue and the known involvement of CaM in diverse cellular processes, was only minimally evident in sustentacular cells, gland cells or respiratory epithelium. Following bulbectomy CaM mRNA declines in the olfactory neuroepithelium as does olfactory marker protein (OMP) mRNA. In contrast to the latter, CaM mRNA makes a partial recovery by one month after surgery. These results, coupled with those from in situ hybridization, indicate that CaM mRNA is expressed in both mature and immature olfactory neurons. The program regulating CaM gene expression in olfactory neurons is distinct from those controlling expression of B50/GAP43 in immature, or OMP in mature, neurons respectively.

Studies on dilution inactivation of sheep liver pyruvate carboxylase.

When sheep liver pyruvate carboxylase was diluted below 4 EU/ml, it underwent inactivation involving two kinetically distinct processes, i.e., a rapid initial burst followed by a slower second phase. The catalytic activity of the diluted enzyme eventually approached zero, suggesting the occurrence of an irreversible process. Analysis of the quaternary structure of the enzyme by gel filtration chromatography and electron microscopy showed that most of the enzyme molecules occur as tetramers at high enzyme concentrations. However, dilution of the enzyme below 4 EU/ml led to the appearance of dimers and monomers which were essentially inactive under the conditions of the assay system used. The presence of acetyl-CoA during dilution prevented inactivation from occurring and preserved the tetrameric structure. When added after dilution, acetyl-CoA prevented further inactivation from occurring but did not reactivate the enzyme. However, acetyl-CoA did cause a relatively rapid reassociation of the inactive monomers and dimers to form inactive tetramers.

Neuroplasticity in the olfactory system: differential effects of central and peripheral lesions of the primary olfactory pathway on the expression of B-50/GAP43 and the olfactory marker protein.

The regeneration of the olfactory neuroepithelium following olfactory bulbectomy or peripheral deafferentation was studied with mRNA probes and antibodies for B-50/GAP43 and for olfactory marker protein (OMP). Two stages in the regeneration of the olfactory epithelium could be discerned with these reagents. The first stage occurs following either peripheral deafferentation of the olfactory epithelium with Triton X-100 (TX-100) or after bulbectomy and is characterized by the formation of a large population of immature olfactory receptor neurons. These newly formed neurons express B-50/GAP43, a phosphoprotein related to neuronal growth and plasticity. During the second stage of the regeneration process the newly formed olfactory neurons mature, as evidenced by a decrease in their expression of B-50/GAP43 and an increase in the expression of OMP. This stage is only manifested if the developing neurons have access to the target olfactory bulb. Formation of a full complement of OMP-expressing neurons occurs only after peripheral lesion with TX-100. In contrast, following bulbectomy the reconstituted olfactory epithelium lacks its normal target and is compromised in its ability to recover from nerve damage, as evidenced by the presence of a large number of B-50/GAP43-expressing neurons up to 3 months after the lesion and its failure to establish a full complement of OMP-expressing neurons. These results demonstrate that the olfactory epithelium is capable of replacing its sensory neurons independently of the presence of its target, the olfactory bulb. However, the differential patterns of expression of B-50/GAP43 and OMP at long times after peripheral lesion with TX-100 or bulbectomy illustrate the profound effect the olfactory bulb has on neuronal maturation in reconstituted olfactory neuroepithelium.

Molecular cloning and sequencing of a cDNA for olfactory marker protein.

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.