James B. Mahony

CHARACTERIZATION OF POLY(A) SEQUENCES IN BRAIN RNA

—Nuclear and polysomal brain RNA from the rabbit bind to Millipore filters and oligo(dT)‐cellulose suggesting the presence of poly(A) sequences. The residual polynucleotide produced after RNase digestion of 32P pulse‐labelled brain RNA is 95% adenylic acid and 200‐250 nucleotides in length. After longer isotope pulses the polysomal poly(A) sequence appears heterodisperse in size and shorter than the nuclear poly (A). Poly(A) sequences of brain RNA are located at the 3′‐OH termini as determined by the periodate‐[3H]NaBH4 labelling technique. Cordycepin interferes with the processing of brain mRNA as it inhibits in vivo poly(A) synthesis by about 80% and decreases the appearance of rapidly labelled RNA in polysomes by about 45%. A small poly(A) molecule 10‐30 nucleotides in length is present in rapidly labelled RNA. It appears to be less sensitive to cordycepin than the larger poly(A) and is not found in polysomal RNA.

Colchicine inhibits the accumulation of messenger RNA for a brain-specific protein in rat glial cells

Abstract The previously reported inhibition of S100 protein synthesis in stationary cultures of C6 cells by antimicrotubular drugs was studied further by assaying the capacity of poly A + RNA from colchicine-treated cultures to direct the synthesis of S100 protein in a wheat embryo cell-free extract. The inhibition of the intracellular S100 protein synthesis by colchicine was paralleled by a decrease in the activity of S100 protein mRNA, suggesting an involvement of the microtubular network in the control of transcription of specific genes.

The Structural Conservation of S100 Protein During Evolution: Analysis by Reactivity with a Monoclonal Antibody

Abstract: A hybridoma cell line producing a monoclonal antibody (A4) against bovine S100 protein has been produced by fusing mouse myeloma P3X63/Ag8 cells with spleen cells from a BALB/c mouse immunized with bovine S100 protein. A4 is of the IgG2b subclass and was purified by affinity chromatography on a protein A‐Sepharose column. Brain extracts from several mammalian and one avian species reacted both with polyclonal rabbit anti‐S100 protein antiserum and with A4 in a radio‐immunoassay. Brain extract from dog was a notable exception. It reacted with the rabbit antiserum but not with A4. Therefore A4 reacts with a common epitope that is present on S100 proteins from different vertebrate species but is absent on dog S100 protein.

Fate of mRNA following disaggregation of brain polysomes after administration of (+)-lysergic acid diethylamide in vivo.

Intravenous injection of (+)-lysergic acid diethylamide into young rabbits induced a transient brain-specific disaggregation of polysomes to monosomes. Investigation of the fate of mRNA revealed that brain poly(A+)mRNA was conserved. In particular, mRNA coding for brain-specific S100 protein was not degraded, nor was it released into free ribonucleoprotein particles. Following the (+)-lysergic acid diethylamide-induced disaggregation of polysomes, mRNA shifted from polysomes and accumulated on monosomes. Formation of a blocked monosome complex, which contained intact mRNA and 40-S plus 60-S ribosomal subunits but lacked nascent peptide chains, suggested that (+)-lysergic acid diethylamide inhibited brain protein synthesis at a specific stage of late initiation or early elongation.

Analysis of Messenger RNA Coding for S100 Protein in the Mammalian Brain

S100 protein is a brain‐specific protein which is absent at birth and first appears in rabbit brain 2–3 days after birth. To determine how the synthesis of this brain‐specific protein is regulated, mRNA was isolated from brain polysomes and assayed for S100 protein mRNA activity by in vitro translation in a heterologous cell‐free system and immunoprecipitation of released polypeptides with rabbit anti‐S I00 protein antiserum. 5100 protein mRNA was detected primarily in small polysomes containing five to eight ribosomes, and virtually no S 100 protein mRNA was present in polysomes containing more than eight ribosomes. S100 protein mRNA was not detected in brain polysomes at stages prior to the induction of synthesis of S100 protein, i.e., in fetal brain or in 1‐day neonates. The amount of S100 protein mRNA in polysomes of the cerebral cortex and cerebellum was measured to see if it correlated with the level of S100 protein in the two regions of adult brain. The cerebellum, which contained three to four times the level of S100 protein in the cerebral cortex, contained four times more S100 protein mRNA.

Selective action of colchicine on protein synthesis and release in a clonal line of rat glial cells

The effect of colchicine on protein synthesis and secretion in stationary cultures of clonal rat glial cells C6 was examined. Colchicine inhibited the synthesis of the brain specific S100 protein in intact cells but not in a cell-free protein synthesizing system derived from these cells. There was no demonstrable effect of the drug on the synthesis of any of the several hundred proteins resolved by a two-dimensional electrophoretic analysis. However, colchicine specifically enhanced the secretion of several proteins of molecular weighs of 30,000 and of 200-300,000 into the medium. Two of the high molecular weight proteins were apparently membrane proteins whose release into the medium was stimulated by the drug.

The synthesis of the brain specific S100 protein in colcemid resistant mutants of rat glial cells

We have isolated two colcemid-resistant mutant sublines, CMR (7A) and CMR (7B), from rat glial cells, C6, using multiple consecutive selections with increasing concentrations of colcemid. The mutant sublines show a decreased uptake of [3H]colchicine but have no apparent defect in the cytoplasmic binding of the drug. The synthesis of the brain-specific S100 protein is less sensitive to colcemid inhibition in the mutant cell lines than in parental C6 cells, suggesting that colcemid must enter the cell to inhibit S100 protein synthesis.

CHARACTERIZATION OF BRAIN RIBONUCLEOPROTEIN PARTICLES

Abstract— Brain RNP particles were characterized to determine whether they play a role in the regulation of brain protein synthesis. RNP particles were isolated from the postribosomal supernatant of cerebral hemispheres of young rabbits, employing conditions which minimize adventitious protein‐RNA interactions. Brain RNP particles consist of a different set of proteins compared to proteins associated with either 40 and 60s ribosomal subunits or polysomal mRNA. Poly(A+)mRNA from brain RNP particles stimulates the incorporation of [35S]methionine in a wheat embryo cell‐free system and codes for a different set of proteins compared to poly(A+)mRNA isolated from polysomes (with some overlap; i.e. mRNA coding for brain‐specific S100 protein is present in both RNP particles and polysomes).

Synthesis of a brain-specific protein (S100 protein) in a lectin-resistant mutant of a rat glial cell line (C6)

The synthesis of S100 protein increases toward the end of the exponential phase of growth of clonal rat glial cells C6 in monolayer culture. Moreover the synthesis of this protein can be increased by treatment of C6 cells with the lectin succinylated concanavalin A (succinyl ConA). In order to study the relationship between these two inductions of S100 protein we have isolated a cell line resistant to ConA from a population of C6 cells. The resistant cells (C6-ConAR) have less succinyl ConA receptors than C6 cells. In contrast to C6 cells, the synthesis of S100 protein does not increase in C6-ConAR cells after treatment with succinyl ConA. However in both cell types the synthesis of S100 protein increases toward the end of the exponential phase of growth. These results suggest firstly that the induction of S100 protein in C6 cells by succinyl ConA is mediated by an interaction of the lectin with its membrane receptors and secondly that the initial steps in the induction of S100 protein by the lectin are different from the initial steps in the induction of this protein which occurs toward the end of the exponential phase of growth in monolayer culture.