Brain Polysomes Research Articles

Expression of Myelin Basic Protein Genes in Several Dysmyelinating Mouse Mutants During Early Postnatal Brain Development

Northern blot and "dot" blot analyses using a myelin basic protein (MBP) specific cDNA probe and in vitro translation techniques were utilized to estimate the relative levels of myelin basic protein messenger RNA (mRNA) in the brains of C57BL/6J control mice, three dysmyelinating mutants (qk/qk, jp/Y, and shi/shi), and three heterozygote controls (qk/+, jp/+, and shi+) during early postnatal development. In general, the MBP mRNA levels measured directly by Northern blot and "dot" blot analyses correlated well with the indirect in vitro translation measurements. The Northern blots indicated that the size of MBP mRNAs in quaking and jimpy brain polysomes appeared to be similar to controls. Very low levels of MBP mRNAs were observed in shi/shi brain polyribosomes throughout early postnatal development. Compared to C57BL/6J controls, accumulation of MBP mRNAs in qk/qk and qk/+ brain polyribosomes was delayed by several days. That is, whereas MBP mRNA levels were below normal between 12 and 18 days, normal levels of message had accumulated in both qk/qk and qk/+ brain polyribosomes by 21 days. Furthermore, normal levels of MBP mRNAs were observed to be maintained until at least 27 days. MBP mRNA levels remained well below control levels in jp/Y brain polyribosomes throughout early postnatal development. The levels did, however, fluctuate slightly and peaked at 15 days in both jp/Y and jp/+ brains, 3 days earlier than in normal mice. Thus, it appears that jimpy and quaking mice exhibit developmental patterns of MBP expression different from each other and from C57BL/6J control mice.

Effects of phenylalanine loading on protein synthesis in the fetal heart and brain of rat: an experimental approach to maternal phenylketonuria.

Pregnant rats were loaded with L-phenylalanine, and the distributions of [14C]leucine and [14C]urea into fetal plasma and tissues were examined. Uptake of [14C]leucine into the supernatant and protein fractions of fetal plasma and tissues was low in the rats loaded with phenylalanine. In contrast, [14C]urea was distributed identically in both groups, indicating that maternal hyperphenylalaninemia did not affect blood flow across the placenta. Administration of phenylalanine and p-chlorophenylalanine produced amino acid imbalance in fetal tissues. Along with these changes, polysomes of the affected fetal heart and brain disaggregated without changes in the ribonuclease activity. These results indicate that high phenylalanine levels in maternal plasma disturb the active transport of amino acids across the placenta, causing an amino acid imbalance and disaggregation of polysomes in fetal heart and brain. These changes may contribute to the congenital heart disease and mental retardation of maternal phenylketonuria.

Effects of trichothecenes (T-2 toxin) on protein synthesis in vitro by brain polysomes and messenger RNA

1. The effects of T-2 toxin on protein synthesis were tested in two reticulocyte lysate In vitro systems pretreated with micrococcal nuclease. One of the test systems contained purified globin mRNA and was initiation dependent. The other contained rat brain polysomes and incorporated amino acids by an elongation dependent process. 2. T-2 toxin inhibited the translation of globin mRNA at all concentrations tested, from 10 −8M to 10 −4M. Rat brain polysomes were much less sensitive to T-2 toxin than globin mRNA. While high concentrations of the toxin (10 −4 M ) led to partial inhibition of protein synthesis by polysomes, low concentrations (10 −8 M and 10 −6 M) stimulated protein synthesis. 3. Comparison of the above results with those obtained by other workers suggests that the T-2 toxin may inhibit not only the initiation step of translation, but also elongation and termination, depending upon the concentration of the toxin and the nature of the translation system. A similar mechanism may operate for all the trichothecene toxins that exert their effect through binding to ribosomal peptidyl transferase.

Purification of γ-enolase messenger ribonucleic acid from rat brain by an immunoadsorption method

Messenger ribonucleic acid (mRNA) coding for the brain-specific protein γ-enolase was isolated by an immunopurification procedure. Rat brain polysomes including nascent polypeptide chains were reacted with specific γ-enolase antibody. The polysome-antibody complexes were subsequently adsorbed to protein A-Sepharose. After extensive washing, RNA ws eluted and applied to an oligo(dT)-cellulose column. Purified mRNA was translated in vitro in a mRNA-dependent rabbit reticulocyte lysate system. The synthesized product was identical to γ-enolase synthesized by free polysomes from rat brain. Immunoisolated γ-enolase mRNA was enriched 380-fold compared to total mRNA extracted from free polysomes. This result indicates that low-abundance mRNAs may conveniently be isolated from brain tissue by immunoadsorption of polysomes.

Induction of a ‘stress’ protein in intact mammalian organs after the intravenous administration of sodium arsenite

The profile of nascent proteins synthesized in various rabbit organs after the intravenous injection of sodium arsenite was analyzed by the cell-free translation of purified polysomes. Examination of the translation products of polysomes isolated 1 hr after injection of sodium arsenite revealed a marked induction of synthesis of a protein of molecular weight 74,000 (74K) in the kidney, heart and liver which was similar to a ‘heat shock’ protein which was induced in these organs after elevation of body temperature by 2·5 to 3°C. Synthesis of the 74K protein was not detected in the translation products of brain polysomes isolated 1 hr after sodium arsenite injection.

Colchicine binding activity of rat brain polysomes

In this communication, we report the presence of a unique colchicine-binding activity in the polysomes of rat brain. This drug-binding property, is somewhat similar to that of tubulin isolated from many sources; however, it differs in several bio-chemical characteristics such as (i) thermal stability of colchicine-binding site, (ii) protection of binding site by vinblastine and (iii) time required for binding equilibration. Such binding of colchicine to the polysomes is most probably due to the presence of a nascent peptide chain of tubulin in the polysome.

Synthesis of S100 protein on free and membrane-bound polysomes of the rabbit brain.

Free and membrane-bound polysomes were isolated from the cerebral hemispheres and cerebellum of the young adult rabbit. The two polysomal populations were translated in an mRNA-dependent cell-free system derived from rabbit reticulocytes. Analysis of the [35S]methionine-labeled translation products on two-dimensional polyacrylamide gels indicated an efficient separation of the two classes of brain polysomes. The relative synthesis of S100 protein by free and membrane-bound polysomes was determined by direct immuno-precipitation of the cell-free translation products in the presence of detergents to reduce nonspecific trapping. Synthesis of S100 protein was found to be twofold greater on membrane-bound polysomes compared with free polysomes isolated from either the cerebral hemispheres or the cerebellum. In addition, the proportion of poly-(A+)mRNA coding for S100 protein was also twofold greater in membrane-bound polysomes compared with free polysomes isolated from the cerebral hemispheres. These results indicate that the cytoplasmic S100 protein is synthesized predominantly on membrane-bound polysomes in the rabbit brain. We suggest that the nascent S100 polypeptide chain translation complex is attached to the rough endoplasmic reticulum by an ionic interaction involving a sequence of 13 basic amino acids in S100 protein.

Translation of mRNA associated with monosomes and residual polysomes following disaggregation of brain polysomes by LSD and hyperthermia.

Intravenous administration of LSD to young adult rabbits induces a transient disaggregation of brain polysomes and a relocalization of mRNA from polysomes to monosomes. To analyze the spectrum of mRNA molecules which were associated with either the residual polysomes or the translationally inactive monosome complex, these two fractions were isolated on sucrose gradients and translated in a reticulocyte cell-free system. Analysis of [35S]methionine labeled translation products by one and two dimensional gel electrophoresis revealed that a full spectrum of mRNA molecules was relocalized from polysomes to monosomes following drug induced polysome disaggregation. The only exception was the mRNA coding for the LSD-induced 74K protein which was associated with the residual polysome fraction and not with the monosome complex. This brain protein is similar in molecular weight to one of the major 'heat shock' proteins which are induced in tissue culture cells following elevation of ambient temperature and disaggregation of existing polysomes. The mRNA coding for the 74K brain protein was not observed in polysomes isolated following blockage of LSD-induced hyperthermia but it was noted when hyperthermia was induced by elevation of ambient temperature. The mRNA species coding for the 74K protein was polyadenylated.

Changes in in vivo rates of protein synthesis on free and membrane-bound polysomes in rat brain during the development of physical dependence on ethanol and after the withdrawal of ethanol

Administration of ethanol and nutrients to rats thrice daily by gavage for 3 days produced a linear increase in physical dependence during the first 3 days and a 2% increase in body weight. Rates of protein synthesis on free and membrane-bound polysomes in whole brain and in 7 brain regions, comprising the entire brain, were measured in vivo under pool expansion conditions with [ 3H]leucine at intervals during the development and decay of ethanol dependence. During dependence development there was a progressive decrease in the rate of protein synthesis on free polysomes, but this change was not significant (P < 0.05) until the third day, and a decrease in the rate on membrane-bound polysomes after 3 days. The inhibition of protein synthesis is attributable to a decreased rate of polypeptide elongation. There was no change in brain weight, DNA content, ribosome content, ribosome distribution on mRNA size. There was, however, a decrease in leucine uptake after 3 days. In an attempt to distinguish between the acute effects of ethanol on regional rates of protein synthesis and those changes associated with dependence development, rates were measured 1.5 h after administering a 5 g/kg dose of ethanol to both control and dependent rats. Rates on free polysomes in the hippocampus-amygdala and thalamus-hypothalamus and on membrane-bound polysomes in the cerebellum and hipocampus-amygdala of dependent rats were reduced; however, there was a general reduction in the rates in control rats that may have obscured reductions in other regions from dependent rats. During early withdrawal, 12 h after the last dose of ethanol, there was an increase in the rate on free polysomes in the pons-medulla and striatum-septum and on membrane-bound polysomes in the hippocampus-amygdala, and a decrease in the rate on free polysomes in the cortex and thalamus-hypothalamus and on membrane-bound polysomes in the cortex. After 24 h, there was an increase in the rate on free polysomes in all regions (cerebellum, cortex, mesencephalon, striatum-septum and thalamus-hypothalamus) except the hippocampus-amygdala and pons-medulla and an increase in the rate on membrane-bound polysomes in all regions (cortex, hippocampus-amygdala, mesencephalon, pons-medulla and striatum-septum) except the cerebellum and thalamus-hypothalamus. The possible relationship of these changes to the homeostat hypothesis of ethanol dependence is discussed.

Blocked and methylated 5'-terminal cap structures of rat brain messenger ribonucleic acids.

The presence and identity of 5'-terminal cap structures in rat brain polysomal mRNA were investigated by radiolabeling the mRNA by periodate oxidation and [3H]sodium borohydride reduction or by beta-elimination of 5'-terminal nucleoside and incorporation of 32P in the presence of polynucleotide kinase. The labeled mRNAs were digested with nucleases and the cap structures were isolated and identified by chromatographic and electrophoretic procedures The results showed that rat brain mRNAs contained cap 1 and cap 2 structures and no caps of the zero type. The proportion of cap 2 was higher than that of cap 1. Both caps had 7-methylguanosine (m7G) as the 5'-terminal nucleoside, which was linked to the next nucleoside by an inverted triphosphate bridge, as in other eukaryotic mRNAs. The most prominent nucleoside in the 5'-penultimate position was 6-methyl-2'-O-methyladenosine [m6A(m)] followed by 2'-O-methyladenosine [A(m)], which together contributed to nearly 70% of both cap 1 and cap 2 structures. 2'-O-Methylguanosine [G(m)] accounted for approximately 18%, the rest being made up of 2'-O-methylcytidine [C(m)] and 2'-O-methyluridine [U(m)].

Comparison of the Effect of Intravenous Administration of d‐Lysergic Acid Diethylamide on Free and Membrane‐Bound Polysomes in the Rabbit Brain

The intravenous administration of LSD to young adult rabbits resulted in the disaggregation of both free and membrane-bound classes of brain polysomes. Based on the analysis of LSD dosage and the time course of the LSD-induced brain polysome shift, it was found that free polysomes were more sensitive to the drug than the membrane-bound polysome fraction. LSD-induced hyperthermia may be involved in the disaggregation of free and membrane-bound polysomes, since a correlation was found between the extent of LSD-induced hyperthermia and the degree of brain polysome shift. Prevention of LSD-induced hyperthermia by maintaining the animal at 4 degrees C blocked the disaggregation of both polysome classes. Induction of hyperthermia by elevation of ambient temperature also resulted in a shift in free and membrane-bound polysomes. In all cases the disaggregation of polysomes to monosomes was not caused by RNase activation. During polysome disaggregation, polyadenylated mRNA associated with both free and membrane-bound polysomes was not degraded but was relocalized from polysomes to monosomes.

In vitro synthesis of human brain proteins including tubulin and actin by purified postmortem polysomes.

Polysomes were prepared from human brain tissue 2-6 h postmortem; the polysomes were active in a cell-free protein synthesis system containing rabbit reticulocyte factors. Protein synthesis was totally dependent upon added MgCl2, ATP, the reticulocyte factor fraction, and the human polysome fraction. Human brain proteins synthesized in the presence of L-[35S]methionine were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis. Over 250 proteins were synthesized and they extended in size up to 250,000 d; many of the most abundant native human brain proteins were synthesized, including tubulin and actin. It was shown that human brain alpha and beta tubulin and actin isomers synthesized in vitro from human postmortem polysomes have the same apparent molecular weights and isoelectric points as the corresponding proteins synthesized by rat polysomes from fresh cortices. The corresponding tubulin and actin synthesized by human and rat brain polysomes also yield the same radioactive methionine-containing peptides after digestion with Staphylococcus aureus V8 protease. These analyses indicate that postmortem polysomes contain active messenger RNA which can direct the partial and/or complete synthesis of actin and tubulin subunits and other human brain proteins.

Cell-free translation of free and membrane-bound polysomes and polyadenylated mRNA from rabbit brain following administration of d-lysergic acid diethylamide in vivo.

Free and membrane-bound polysomes and polyadenylated mRNA isolated from rabbit brain were translated in an mRNA-dependent rabbit reticulocyte lysate system. Electrophoretic analysis of the cell-free translation products demonstrated that although most of the nascent proteins were common to both free and membrane-bound brain polysomes, qualitative and quantitative differences were observed. Compared with the results obtained with purified polyadenylated mRNA, the addition of intact polysomes to the cell-free translation assay was more efficient and produced higher molecular weight products. Analysis of the translation products of free and membrane-bound polysomes revealed the appearance of 74K protein following neither LSD administration or hyperthermia induced by elevated temperature treatment. The presence of this 74K protein was verified by analysis of the translation products by two-dimensional gel electrophoresis.

Effect of Electroconvulsive Shock on Polysomes of Rabbit Brain, Liver, and Kidney

Following electroshock treatment of the rabbit, polysomes have been found to disaggregate in the liver and kidney to an extent comparable with disaggregation in the cerebral cortex. After a single electroconvulsive shock, the process of polysomal disaggregation in the former two organs reaches maximum values at 10 and 60 min, but returns to control values at intermediate times. In the cerebral cortex the shift to control values observed at 20 min is not statistically significant.

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-S100 protein antiserum. S100 protein mRNA was detected primarily in small polysomes containing five to eight ribosomes, and virtually no S100 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.

Identification of rat brain polysomes synthesizing the brain specific enolase (14.3.2 protein), S100 protein and α and β tubulin subunits

Polysomes prepared from frozen rat brain powder were fractionated by centrifugation in a sucrose gradient. Individual fractions were used to program a reticulocyte lysate in a runoff reaction. The products of cell-free synthesis were assayed for the brain-specific enolase (14.3.2 protein) and S100 protein by immunoprecipitation with specific antisera and for tubulin by two-dimensional electrophoresis in polyacrylamide slab gels. The relative synthesis of these proteins by unfractionated free brain polysomes were 0.1 per cent, 0.05 per cent and 0.7 per cent respectively. After centrifugation in a sucrose gradient polysomes synthesizing S100 protein were separated from those synthesizing the other two markers. There was a threefold enrichment in the specific messenger RNA activity for each of the three proteins studied in their respective peak fractions of polysomes.

Comparison of nuclear and polysomal RNA fractions from goat brain

Phenol extracted RNA preparations from highly purified nuclei and polysomes of goat brain were fractionated by chromatography on oligo (dT)-cellulose and analyzed by electrophoresis on agarose-acrylamide composite gels. The electrophoretic profile of the polysomal polyadenylated RNA fraction showed a major band with a molecular weight of about 0.62 × 10 6, which corresponds to the size of the tubulin mRNA. The nuclear polyadenylated RNA fraction also displayed a single major band, with an estimated molecular weight of 0.76 × 10 6, which appears to be a potential precursor of tubulin mRNA.

Disaggregation of brain polysomes after LSD in vivo. Involvement of LSD-induced hyperthermia.

LSD-induced hyperthermia is implicated in the brain-specific disaggregation of polysomes which is induced following intravenous administration of the drug to rabbits. Both LSD-induced hyperthermia and brain polysome disaggregation were found to increase in parallel under conditions which accentuated the effect of the drug on brain protein synthesis. Pretreatment with neurotransmitter receptor blockers or placing the animal at an ambient temperature of 4 degrees C after LSD administration prevented both hyperthermia and brain polysome disaggregation. The administration of apomorphine, which causes hyperthermia in rabbits also caused disaggregation of brain polysomes. Direct elevation of the body temperature to levels similar to that found after LSD was achieved by placing animals at an ambient temperature of 37 degrees C. Under these conditions a brain-specific disaggregation of polysomes resulted which was not due to RNAase activation. After either LSD or direct heating, the brain polysome shift was associated with a relocalization of polyadenylated mRNA from polysomes to monosomes as determined by [3H]polyuridylate hybridization. Since polysome disaggregation was found only in brain, it appears that the brain may be more sensitive to elevations in body temperature compared to other organs.

Complex population of nonpolyadenylated messenger RNA in mouse brain

The complexity of nonadenylated mRNA [poly(A)-mRna]has been determined by hybridization with single-copy DNA (scDNA) and cDNA. Our results show that poly(A) − and poly(A) + mRNA are essentially nonoverlapping (nonhomologous) sequence populations of similar complexity. The sum of the complexities of poly(A) + mRNA and poly(A) − mRNA is equal to that of total polysomal RNA or total mRNA, or the equivalent of ∼1.7 × 10 5 different sequences 1.5 kb in length. Poly(A) − mRNA, isolated from polysomal RNA by benzoylated cellulose chromatography, hybridized with 3.6% of the scDNA, corresponding to a complexity of 7.8 × 10 4 different 1.5 kb sequences. The equivalent of only one adenosine tract of ∼20 nucleotides per 100 poly(A) − mRNA molecules 1.5 kb in size was observed by hybridization with poly(U). cDNA was transcribed from poly(A) − mRNA using random oligonucleotides as primers. Only 1–2% of the singlecopy fraction of this cDNA was hybridized using poly(A) + mRNA as a driver. These results show that poly(A) −mRNA shares few sequences with poly(A) + mRNA and thus constitutes a separate, complex class of messenger RNA. These measurements preclude the presence of a complex class of bimorphic mRNAs [that is, species present in both poly(A) + and poly(A) − forms]in brain polysomes.

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.