Lawrence D. Grouse

SEQUENCE DIVERSITY STUDIES OF RAT BRAIN RNA: EFFECTS OF ENVIRONMENTAL COMPLEXITY ON RAT BRAIN RNA DIVERSITY

The sequence complexities of rat brain RNAs were measured by RNA‐driven hybridization reactions with nonrepetitive rat DNA. The total sequence complexity of rat brain HnRNA was estimated to be 6.61 x 108 nucleotides while rat brain poly(A)‐mRNA sequence complexity was 1.32 x 108 nucleotides. Up to 33.7% of the total transcribable nonrepetitive DNA was expressed in the nuclear RNA. The nuclear RNAs reacted with complex kinetics over at least 4.5 decades of equivalent Rot (product of RNA concentration and time), with an apparent division into three major RNA abundance classes. The abundances of average nuclear RNA species in these classes ranged from 2.9 x 109 copies per brain (18 copies per cell) to 2.4 x 105 copies per brain (1.5 x 10−3 copies per cell). Poly(A)‐mRNA diversity was sufficient to code for 8.8 x 104 polypeptides of 50,000 daltons. There were also three distinguishable abundance classes of poly(A)‐mRNA with frequencies which ranged from 8.9 x 108 copies per brain (5.5 copies per cell) to 3.2 x 105 copies per brain (2 x 10−3 copies per cell). Evidence for compartmentalization of expressed RNA sequences supports the concept that the extensive morphological and physiological specialization evident in brain parallels extensive transcriptional specialization at the cellular level.

BRAIN NUCLEIC ACIDS

Publisher Summary This chapter discusses the content of nucleic acids in the brain. The DNA content of an adult rat brain is about 0.7 to 1.0 mg per gram of fresh tissue. 98% percent or more is located in the nuclei, with the remaining 1–2% being mitochondrial. As the normal DNA content of a mammalian diploid cell is about 6.5 pg, the DNA content is more frequently used to estimate the number of cells in a given tissue under specific conditions or at a certain developmental stage. During postnatal growth the cytoplasmic volume increases, the space between the nuclei becomes greater, and the DNA content per unit of wet weight decreases, even though the number of cells remains constant or even increases. The RNA content of fresh cortical tissue of rats is about 0.7–0.8 mg per gram of tissue. Thus, the RNA/DNA ratio of brain tissue is about 1.0. Some evidence suggests that the concentration of RNA in oligodendrocytes is higher than that in neurons and that astrocytes contain a low RNA concentration. The great preponderance of the RNA in brain, as in all eukaryotic tissues, is of the ribosomal type. There are no unusual types of RNAs in brain; the general characteristics of the ribosomal, transfer RNA, and messenger RNA fractions are much like those of the corresponding RNAs from other tissues.

Effect of visual experience on gene expression during the development of stimulus specificity in cat brain

Abstract The eyelids of newborn kittens were bilaterally sutured in an experiment to test the effect of patterned visual experience on genetic transcription. A comparison was made of total RNA complexity between brain RNAs from kittens with sutured eyelids and those of their unsutured littermates. The complexity of visual cortex RNA from normally sighted animals was greater than that of lid-sutured animals. In contrast, RNAs from total nonvisual cortices of sutured and unsutured kittens contained indistinguishable RNA sequence complexities, as did RNAs from subcortical structures. Thus, the normal maturation of visual cortex which is dependent on visual experience appears to involve a greater amount of genetic expression than occurs in the absence of visual experience.

chapter 12 Rna Sequence Complexity in Central Nervous System Development and Plasticity

Publisher Summary Several lines of research with brain RNA have developed since the initial RNA complexity studies began. This chapter assesses the present understanding of brain transcription, with reference to hybridization studies with nonrepetitive and complementary DNA. Recent advances in the understanding of RNA processing and its relation to brain RNA are reviewed. The chapter discusses the studies showing effects of development, aging, and sensory-environmental factors on gene expression. The conclusions of studies on the RNAs of clonal cell lines of neural origin are presented, and the implications of these data for characterization of the transcriptional pattern in brain are also discussed. Unlike studies focusing on the expression of individual gene products in brain or on the specific physiological properties of nerve cells, studies on the expression of nonrepetitive DNA are able to define the total transcriptional or translational response of the brain to such key neurobiological events as differentiation and synapse formation.

Radioactive labeling of DNA in cells in culture.

Abstract We have compared and evaluated various methods for recovery and deproteinization of DNA from cells in culture and for obtaining labeled DNA at high specific activities with [ 3 H]thymidine. The kinetics of labeling cells in culture with [ 3 H]thymidine were examined using the Chang human liver cell line. The extent of labeling was limited by the presence of the radioactive nuclides in the intracellular pool, and the limitation was associated with cessation of cell multiplication after approximately one doubling. Incorporation of [ 3 H]thymidine was found to be most cost-efficient at low cell densities and at a precursor concentration of 3 μCi/ml in the presence of 5-fluorodeoxyuridine; however, higher precursor concentrations led to greater DNA specific activity. The extent of label incorporation varied with different vertebrate cell lines tested.

Measurements of gene expression in tissues of normal and dystrophic mice.

Abstract RNA sequence complexities of normal and dystrophic (dy 2J /dy 2J ) mouse tissues were measured to determine whether or not the disease process altered the transcriptional diversity patterns of normal development. We found that dystrophic muscles contained a 60% higher concentration (per milligram of total RNA) of complex, low-frequency RNA species than did control muscles, although the complexities of dystrophic and control preparations were not distinguishable. Total cellular RNAs from spinal cords, brains, and livers of dystrophic mice were also found to have RNA complexities which were indistinguishable from the same tissues in normal mice. Electrophoretic studies of several muscle-specific isoenzymes in normal and dystrophic muscle showed no evidence of the expression of alternate isoenzymes in dystrophic muscle. From these results we concluded that a block in differentiation was not likely to be the cause of the disease.

Sequence complexity and frequency distribution of poly(A)-containing messenger RNA sequences from the glioma cell line (C6.

OUR CONCEPT of gene transcription has undergone important changes over the past few years, particularly as a result of recent data obtained with recombinant DNA technology (GARAPIN et a/., 1978; LAI et al., 1978). Recent work on nuclear RNA suggests that processing of a nuclear RNA molecule yields a mature messenger RNA (mRNA) molecule (TILGHMAN et al., 1978). Since the most extensive gene expression in any mammalian organ occurs in the brain, where as much as 32% of all potential genes may be expressed in nuclear RNA (BANTLE & HAHN, 1976; BROWN & CHURCH, 1972; CHIKARAISHI e t al.. 1978; GROUSE et al., 1972, 1978; HAHN & LAIRD, 1971), it follows that if all mRNAs arise from nuclear RNA processing, 32% of all possible mRNAs would also be expressed in brain. The mRNA complexity of brain appears to be widely apportioned among different cell types. Our recent calculations showed (GROUSE et al., 1978) that rat brain can be divided into about 7000 different cell types on the basis of RNA sequence complexity and frequency data. Each of these cell types would be characterized by the expression of 1C-20 specific genes. Little is known, however, about the relative RNA complexities of the neuronal and glial elements of brain. In the absence of highly purified diploid cell types from brain, minimal deviation tumor cells of the nervous system were studied. Our recent data (SCHRIER et al., 1978) on the RNAs of mouse neuroblastoma provide a model of neuronal cell transcription. The nuclear RNA of the neuroblastoma clone S20:DB6 bad a sequence complexity of 9 x lo7 nucleotides, while the complexity of the poly(A)-containing mRNA was 4.4 x 10’ nucleotides. For comparison with these data, the subject of this communication will be the results of our analysis of the complexities and abundances of poly(A) mRNA species in a glial cell line, the cloned rat astrocytoma cell line C6. The cell line C6 has frequently been used in neurobiological studies as a glial cell model. It has been shown to respond to hydrocortisone (BENNETT et al., 1977) and /J-adrenergic agonists (FRANKLIN & TWOSE, 1977; GILMAN & NIRENBERG, 1971), and to have regulatable synthesis of the S-100 protein (LABOURDETTE et a/., 1977), uptake mechanisms for putative neurotransmitters (SCHRIER & THOMPSON, 1974) and specific cell adhesive properties (SANTALA & GLASER, 1977). We used the diploid C6Bul clone of C6, which we obtained from Dr. MARSHALL NWENBERG. This cell line

Effects of estrogen on gene expression in rooster liver.

Abstract The effects of estrogen on RNA sequence complexity and sequence frequency were studied in rooster liver. Both control and estrogen-treated liver contained total RNA sequence diversity of approximately 4.2 × 10 7 nucleotides. Two components were found in the reaction of chicken liver or brain RNA with unique DNA: RNA species present at high concentration and RNA species about 100-fold less abundant. Approximately 7 × 10 6 nucleotides of RNA sequence complexity were present at high concentration in estrogen-treated liver but not at high concentration in control liver.

The use of glucosamine and actinomycin D in radioactive labeling of RNA in cells in culture.

Abstract Pretreatment of confluent cultures of mouse L cells or of well-differentiated nervous system cells in primary cultures with 20–120 mM glucosamine resulted in a stimulation of the uptake of tritiated uridine, but not of adenosine. A marked stimulation of the incorporation of radioactive uridine into acid-precipitable macromolecules was also obtained, while adenosine incorporation was unchanged. Cultures of L cells in log phase of growth were similarly affected by glucosamine pretreatment. Uridine and cytidine uptakes were stimulated by 50%. Tritiated uridine incorporation was stimulated in a biphasic manner, with maximal stimulation (115%) after 15–60 min of labeling and at later times an inhibition of incorporation. The stimulation of cytidine incorporation paralleled the stimulation of its uptake. The data indicate that there is: a) a glucosamine-induced stimulation of pyrimidine nucleoside uptake, b) a marked stimulation of tritiated uridine incorporation into RNA due to depletion of the cellul...