Expression and developmental regulation of two unique mRNAs specific to brain membrane-bound polyribosomes

Abstract

Translation in vitro of membrane-bound polyribosomal mRNAs from rat brain has shown several to be developmentally regulated [Hall & Lim (1981) Biochem. J. 196,[327][328][329][330][331][332][333][334][335][336]. Here we describe the isolation and characterization of cDNAs corresponding to two such brain mRNAs. One cDNA (M444) hybridselected a 0.95 kb mRNA directing the synthesis in vitro of a 21 kDa pl-6.3 polypeptide, which was processed in vitro by microsomal membranes. A second cDNA (M1622) hybridized to a 2.2 kb mRNA directing the synthesis of a 55 kDa pl-5.8 polypeptide. Both mRNAs were specific to membrane-bound polyribosomes. Restriction maps of the corresponding genomic DNA sequences are consistent with both being single copy. The two mRNAs were present in astrocytic and neuronal cultures, but not in liver or spleen or in neuroblastoma or glioma cells. The two mRNAs were differently regulated during brain development. In the developing forebrain there was a gradual and sustained increase in M444 mRNA during the first 3 weeks post partum, whereas M 1622 mRNA appeared earlier and showed no further increase after day 10. In the cerebellum the developmental increase in M444 mRNA was biphasic. After a small initial increase there was a decrease in this mRNA at day 10, coincident with high amounts of M1622 mRNA. This was followed by a second, larger, increase in M444 mRNA, when amounts of M1622 mRNA were constant. The contrasting changes in these two mRNAs in the developing cerebellum are of particular interest, since they occur during an intensive period of cell proliferation, migration and altering neural connectivity. As these mRNAs are specific to differentiated neural tissue, they represent useful molecular markers for studying brain differentiation.

Identification of proteins that are developmentally regulated during early cerebral corticogenesis in the rat

“…In addition, the increase in the rate of tubulin synthesis from E 14 to E2 1 detected in the quantitative analysis of the 2-D fluorograms (see Fig. 9) is in agreement with the results reported by others (Denoulet et al, 1982;Hall et al, 1987).…”

“…As far as we can tell, none of the other developmentally regulated proteins identified in the present analysis have isoelectric points and molecular weights that correspond to other known developmentally regulated proteins in the mammalian CNS (Skene and Willard, 198 1;Bennett et al, 1982;Shaw and Weber, 1982;Dodd et al, 1984;Rathjen and Schachner, 1984;Darmon and Paulin, 1985;Riederer and Matus, 1985;Jacobson et al, 1986;Lenoir et al, 1986;Yamamoto et al, 1986;Baizer and Fishman, 1987;Hall et al, 1987;Miller et al, 1987;Szaro and Loh, 1987;Edmondson et al, 1988;Horton and Levitt, 1988; Figure 12. Some of these developmentally regulated proteins are enriched in brain.…”

Human carboxypeptidase E. Isolation and characterization of the cDNA, sequence conservation, expression and processing in vitro

“…One of these (M1622 cDNA), corresponding to a 2.2 kb mRNA expressed in brain and adrenal gland, can be identified as rat CPE on the basis of its sequence similarity to bovine and rat CPEs (Fricker et al, 1986(Fricker et al, , 1989Rodriguez et al, 1989). Rat CPE mRNA is developmentally regulated in the brain (Hall et al, 1987), being detectable at embryonic day 17 and increasing to maximal concentrations 10 days after birth, concordant with the period of cellular differentiation and establishment of functional synapses. Interestingly, CPE mRNA was expressed in primary glial as well as neuronal cultures (Hall et al, 1987;Lowndes, 1988).…”

“…Rat CPE mRNA is developmentally regulated in the brain (Hall et al, 1987), being detectable at embryonic day 17 and increasing to maximal concentrations 10 days after birth, concordant with the period of cellular differentiation and establishment of functional synapses. Interestingly, CPE mRNA was expressed in primary glial as well as neuronal cultures (Hall et al, 1987;Lowndes, 1988). CPE is a glycoprotein, which is synthesized as a larger inactive precursor (Fricker, 1988).…”

“…Many membrane proteins and peptide precursors are synthesized by polyribosomes bound to membranes of the endoplasmic reticulum, where the first stages of glycosylation and other post-translational processing events occur. In our previous investigations of this class of brain membrane proteins we isolated and characterized two unique developmentally regulated cDNAs (Hall et al, 1987). One of these (M1622 cDNA), corresponding to a 2.2 kb mRNA expressed in brain and adrenal gland, can be identified as rat CPE on the basis of its sequence similarity to bovine and rat CPEs (Fricker et al, 1986(Fricker et al, , 1989Rodriguez et al, 1989).…”