Research report
Differentially expressed olfactomedin-related glycoproteins (Pancortins) in the brain

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Abstract

Messenger RNA differential display is conducted to search for genes that are expressed in a region-specific pattern in the rodent brain. Eleven novel gene fragments are isolated. One of these genes which we call pancortin, based on its predominant mRNA expression in the cerebral cortex of the adult, is studied. These pancortin cDNA clones are grouped into four different types of cDNA, designated as pancortin-1 to -4. All pancortin cDNAs share a common sequence in the middle of their structure, having two alternative sequences at both 5′- and 3′-ends, respectively. Deduced amino acid sequence shows that all pancortins have sequences of hydrophobic amino acids at N-terminus and no obvious membrane spanning regions. In situ hybridization histochemistry using oligonucleotide probes specific for 5′- and 3′-end variable parts has revealed that these four pancortin mRNAs are expressed differentially in the adult rodent brain. Robust expression of pancortin-1 and -2 mRNA is observed in the cerebral cortex (including the hippocampus and the olfactory bulb). However, little of pancortin-3 and -4 mRNA is observed there. In the cortex, some neurons are stained by an antibody raised against Pancortin. Immuno-electron microscopic study has revealed that Pancortin-like immunoreactive products are localized mainly in the endoplasmic reticulum and not in the Golgi apparatus indicating that Pancortins are the endoplasmic reticulum-anchored proteins. Our results suggest that each Pancortin is differentially regulated and may perform different functions in the brain.

Introduction

The central nervous system (CNS) in higher animals is composed of many regions which are both functionally and histologically distinct. What makes one region functionally distinct from others? How different is one region from another? A factor expressed exclusively or predominantly in a specific region appears to be associated with such specificity [3]. A well-known example is the G-protein-coupled receptors, one of which is expressed exclusively in the neurons of the olfactory epithelium and related to olfactory functions [5]. The search for and analyses of genes expressed in a region-specific manner in the CNS are one of the applicable strategies in identifying specific factors or in studying region-specific neuronal functions at the molecular level.

Much effort has been made to search for genes which are expressed in a region-specific manner in the brain. The subtraction method is one strategy used for this purpose 1, 3, 21, 22, 24. Recently, an innovative technique, the messenger RNA (mRNA) differential display method 9, 10, has been developed. This method enables us to compare, identify and acquire genes expressed only under some specific conditions and/or regions 4, 6, 11. With this method, we have been searching for a gene expressed in the pons during the neuronal circuit formation period. One such gene, which we call pancortin, based on its predominant mRNA expression in the cerebral cortex of the adult, is studied in our research.

Section snippets

Animals

Adult female Wistar rats, weighing approximately 300 g, and newborn pups were used. Rats were purchased from a local vendor (Nippon Clea, Japan). They were housed in a room kept at a constant temperature and humidity and were provided with food and water ad libitum. Diurnal lighting conditions were maintained with lights on from 08:00 to 20:00 h. Pups were delivered from housed Wistar rats which were obtained 2 days prior to full-term pregnancy. Pups were used for experiments within 24 h of

Isolation of a gene which is expressed predominantly in the cortex

We isolated 17 gene fragments through several screenings using the mRNA differential display method (see Section 2). Among them, 11 fragments turned out to be novel after sequencing and DNA Data Base inquiry (GenBank, October, 1993) by DNASIS program (Hitachi, Japan). ISHH with macro-autoradiogram was carried out to examine their regional distribution in the brain. Among 11 novel candidates, one DNA fragment designated as #103 showed strong hybridization signals in the cortex of the adult rat (

Discussion

Pancortins consist of four independent members (designated Pancortin-1 to -4) sharing a common part in the center of their sequences.

Acknowledgements

The first two authors (T.N. and A.N.) contributed equally to this work. We thank Ms. Sachiyo Funai for her assistance with the Western blot analysis and Ms. Ikuyo Jikihara for her work with the electron microscope. This work was supported in part by Naito Foundation, Senri-life Science Foundation and the Ministry of Education, Science, Sports and Culture of Japan (to M.S.). The DNA database accession numbers for mouse Pancortin-1 through -4 are: D78262–D78265.

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    1

    Present address: Department of Molecular Neurobiology, Brain Research Institute, Niigata University, Niigata, 951 Japan.

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