INCORPORATION IN VIVO OF RADIOACTIVE LEUCINE INTO NEURONAL AND GOAL NUCLEAR PROTEINS OF RAT BRAIN

Purified neuronal and glial nuclei were separated from rat brain cells. The fraction rich in neuronal nuclei contained 68 ± 9 per cent neuronal nuclei and the fraction rich in glial nuclei contained 89 ± 6 per cent glial nuclei. The fraction rich in neuronal nuclei isolated from cells of adult rat brain incorporated l -[4,5-³H]leucine into TCA-insoluble material at a rate comparable to those of the microsomal and the soluble fractions of the brain, and at a much higher rate than the fraction rich in glial nuclei. The proteins soluble in buffered-saline, the acid-soluble deoxyribonucleoproteins, and the residual proteins of the neuronal nuclei are apparently the proteins which account for the higher specific activity of neuronal proteins compared with glial nuclear proteins. In liver and kidney, the incorporation of [³H]leucine into nuclear proteins was lower than into other subcellular fractions from the same organs.     

Nuclear DNA Ligase and Its Action on Chromatin DNA in Neuronal, Glial and Liver Nuclei Isolated from Adult Guinea Pigs

Abstract: DNA ligase activities were measured in neuron-rich and glial nuclear preparations and liver nuclei isolated from adult guinea pigs. The enzymatic properties of cerebral and liver nuclear DNA ligases were studied with isolated nuclei and nuclear extracts. ATP (K_m= 46–48 μM) and bivalent cation (Mg²⁺ or Mn²⁺) were required for the maximal activities in cerebral and liver nuclei. β-Mercaptoethanol did not affect the activities, but N-ethylmaleimide and p-chloromercuribenzoate completely inhibited the activities. Deoxyadenosine-5′-triphosphate partially inhibited the activities in both cerebral and liver nuclei. An interdependent effect of Na⁺ and Mg²⁺ on the enzyme activities was observed. A high concentration (200 mM) of Na⁺ activated both enzymes and shifted to the acid side the optimal pH for both enzymes. DNA ligase was more easily extracted with lower concentrations of NaCl from liver nuclei than from cerebral nuclei, but the extraction curves from both nuclear species reached a plateau level (92% of total activities of nuclear enzymes) at 200 mM-NaCl. Apparent K_m for the substrate [³²P]phosphoryl DNA was determined according to a modification of the Michaelis-Menten equation, which was applied for the case where an unknown amount of substrate nicks in chromatin DNA coexisted with the nicks in exogenous substrate DNA. Neuronal and glial nuclear enzymes had similar K_m values (about 20 μg of [³²P]phosphoryl DNA/ml), but the liver nuclear enzyme had a higher K_m value (54 μg of [³²P]phosphoryl DNA/ml). The modified Michaelis-Menten equation provided the amounts of nicks available as substrate in chromatin DNA of isolated nuclei. Neuronal and glial nuclei contained 1.5 and 0.29 pmol of nicks/μg of nuclear DNA, respectively, in contrast to an intermediate amount of nicks in liver nuclei (0.63 pmol/μg of nuclear DNA). DNA ligase activity in neuronal nuclei [312 units (fmol of 5′-phosphomonoester converted into a phosphatase-resistant form per min at 37°C) per μg of nuclear DNA] was 11-fold higher than that in glial nuclei [28.7 units/μg of nuclear DNA]. Liver nuclei contained an intermediate activity [54.7 units/μg of nuclear DNA].     

Studies on ribonucleic acid and homopolyribonucleotide formation in neuronal, glial and liver nuclei

1. Various types of nuclear preparations, with different ratios of neuronal to glial nuclei, were isolated from guinea-pig cerebral grey matter and ox cerebral grey matter and white matter. Conditions appropriate for the separate assay of RNA and poly A formation were described. Comparative rates of RNA and poly A formation were studied in cerebral and liver nuclei. 2. RNA polymerase activity per nucleus is higher in neuronal nuclei than in glial nuclei. In liver nuclei, the activity is much lower than in cerebral nuclei. The physical relationship between RNA polymerase and deoxyribonucleoprotein seems to differ in neuronal, glial and liver nuclei. 3. Poly A polymerase activity in liver nuclei is selectively activated by Mn(2+) and inhibited by GTP, CTP and UTP. On a DNA basis, the activity in an aggregate enzyme is the same as in intact nuclei. Poly A polymerase activity per nucleus is much higher in liver nuclei than in neuronal nuclei. Glial nuclei show an intermediate activity. 4. It is suggested that, in neuronal nuclei, the synthesis of RNA is more prominent than that of poly A under conditions where both polymers are formed simultaneously. This contrasts with liver nuclei, where more poly A is made than RNA. 5. In neuronal nuclei, the rate of CTP incorporation is much higher than in glial and liver nuclei. This incorporation is most probably due to poly C synthesis.     

Catalytic properties of DNA polymerase α activity associated with the heat-stabilized nuclear matrix prepared from HeLa S3 cells

We have investigated whether or not ATP or other nucleoside di- and trisphosphates (including some nonhydrolysable ATP analogues) can stimulate the activity and/or the processivity of DNA polymerase α associated with the nuclear matrix obtained from HeLa S3 cell nuclei that had been stabilized at 37°C prior to subfractionation, as has been reported previously for DNA polymerase α bound to the nuclear matrix prepared from 22-h regenerating rat liver. We have found that HeLa cell matrix-associated DNA polymerase α activity could not be stimulated at all by ATP or other nucleotides, a behaviour which was shared also by DNA polymerase α activity that solubilizes from cells during the isolation of nuclei and that is thought to be a form of the enzyme not actively engaged in DNA replication. Moreover, the processivity of matrix-bound DNA polymerase α activity was low (< 10 nucleotides). These results were obtained with the matrix prepared with either 2M NaCl or 0·25 M (NH₄)₂SO₄ and led us to consider that a 37° incubation of isolated nuclei renders resistant to high-salt extraction a form of DNA polymerase α which is unlikely to be involved in DNA replication in vivo.     

Thyroid hormone receptors of developing chick brain are predominantly in the neurons

The relative concentration of the triiodothyronine (T₃) receptors in the neuronal and glial nuclei of developing chick brain have been studied. Scatchard analysis indicate that the number of T₃ binding sites in the neuronal nuclei increases from 400 to 1600 sites/nucleus between 7–11 day of embryonic development without any concomitant change in the level of glial nuclear receptors (130 – 200 sites/nucleus). Both sites are of high affinity (K_a = 1–3 × 10⁹ M^?1) at all ages examined. The abundance of the T₃-receptors in the neuronal nuclei and the close coincidence of the period of rise in the level of these receptors in these nuclei (7–11 day) with that of maximal neuronal growth and synaptogenesis (7–13 day) suggest that the neurons are the primary site of action of T₃ in the developing brain.     

Poly(adenosine diphosphate ribose) polymerase activity in neuronal and glial nuclei from bovine cerebrum

Two different preparations isolated from beef cerebrum have been used to compare the polyadenosine diphosphate ribose (polyADPR) polymerase activities in neuronal and glial nuclei: (1) nuclear suspensions (with or without DNase I treatment), and (2) 1 M NaCl nuclear extracts (soluble enzyme). The DNAse I treatment of nuclei and the solubilization of polyADPR polymerase by 1 M NaCl enhances the polyADPR polymerase activity. The polyADPR polymerase activity is similar in neuronal and glial nuclear suspensions, while the neuronal soluble enzyme activity is significantly higher than that of the glial soluble enzyme. Evidence is presented that the difference in soluble enzyme activities is not due to the effects of DNA or degrading enzymes. Some activating factor(s) seem to be present in neuronal soluble extracts, while both inhibiting and activating factor(s) seem to be present in glial soluble extracts.     

Increased nuclear DNA content in developing cotton fiber cells

The nuclear DNA content of developing cotton fiber cells (Gossypium hirsutum, cv. MD51ne) increases ∼24% after 2 d postanthesis (dpa). The amount of nuclear DNA at 2 dpa is 5.4 ± 0.27 pg. At 3–4 dpa it increases to 6.7 ± 0.24 pg and by 5 dpa it is 6.8 ± 0.70 pg. These values were obtained by nuclear fluorescence after staining with Hoechst 33258. Human oral squamous cell nuclei were used as a DNA standard. Nuclear DNA content increases in fibers growing on either fertilized or unfertilized ovules. The increase also is detectable in Feulgen stained nuclei using two-wavelength cytospectrophotometry. All measurements were made on isolated fiber cell nuclei using a newly developed method tailored to cotton fiber cells. The results imply that during the early stages of development fiber cell nuclei either selectively amplify certain sequences or enter S-phase replicating a portion of their genome.     

Absence of high levels of DNA polymerase α activity in the nuclear matrix prepared from mouse erythroleukemia cells

We have examined the association of DNA polymerase α activity with the nuclear matrix prepared by different techniques from mouse erythroleukemia cells. At variance with the data obtained using other cell types we have found that only a small amount (less than 2%) of nuclear DNA polymerase α activity resisted extraction with high-ionic strength buffers, even if nuclei were heat-stabilized by incubation at 37°C for 45 min prior to subfractionation. The recovery of DNA polymerase α activity bound to the matrix was unaffected by the type of extracting agent used (NaCl or (NH₄)₂ SO₄), by the extraction sequence or by the method employed for obtaining nuclei. These results could indicate that in some types of cells the nuclear matrix is not involved in DNA replication.     

Ligation and synthesis of chromatin deoxyribonucleic acid in vitro in neuronal, glial and liver nuclei isolated from adult guinea pig

Neuron-rich and glial nuclear preparations and liver nuclei were isolated from adult guinea pigs. These nuclei were incubated to carry out DNA-ligation and -synthesis reactions. Before and after incubation, the sizes of single-standed DNA and DNA-synthesis patterns in single strands were analysed by using alkaline sucrose-density-gradient centrifugation. Isolation of nuclei by cell-fractionation technique shortened chromatin DNA and decreased markedly the number-average molecular weight of DNA strands. Chromatin DNA in neuronal and glial nuclei was ligated at the nicks during incubation in a reaction mixture containing ATP, Mg(2+), dithiothreitol and four deoxyribonucleotides. The number-average molecular weights were estimated to increase 1.1-and 2.1-fold in neuronal and glial nuclei respectively. DNA strands in liver nuclei were shortened during incubation, but elongated under conditions that inhibit deoxyribonuclease. Since the endogenous deoxyribounuclease activity was conspicuously higher in liver nuclei than in neuronal and glial nuclei, the shortening and elongation were thought to depend on the balance between DNA ligase and deoxyribonuclease reactions. DNA synthesis occurred at the gaps in chromatin DNA and about 50% of the total synthesized DNA was found in the shorter strands having 6 to 297 bases in all species of nuclei. Based on these results, it was concluded that in nuclei isolated from non-dividing cells (neurons) and slowly dividing cells (glial and liver cells) DNA-ligation and -synthesis reactions proceeded in parallel at the breaks in single-stranded DNA, which was produced mainly by endogenous deoxyribonuclease during isolation and incubation processes.     

Nuclear metabolism of benzo(a)pyrene and of (±)-trans-7,8-dihydroxy-7,8,-dihydrobenzo(a)pyrene: Comparative chromatographic analysis of alkylated DNA

Rat liver nuclei were incubated with [¹⁴C]benzo(a)pyrene (BP) or [³H](±)-trans-7,8-dihydrodiol of BP (³H-BP-7,8-diol) in the presence of a NADPH-generating system. The nuclei were able to form from BP the 9,10-, 4,5- and 7,8-dihydrodiols, the 3,6- and 1,6-quinones as well as the 3- and 9-phenols. The total nuclear metabolism was stimulated 11-fold by prior administration to the rats of 3-methylcholanthrene (3MC). BP-7,8-dihydrodiol formation, under these circumstances, was enhanced 29-fold. The rat liver nuclei were also able to form from [³H]BP-7,8-diol, (±)-7β,8α-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydro BP (diol epoxide 1), (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydro BP (diol epoxide 2), as well as three unknown metabolites. Diol epoxides 1 and 2 represented 23 and 65% of the total metabolites produced during the control nuclear incubation. Pretreatment of the rats with 3MC resulted in 4-fold increase in nuclear metabolic activity. Under the latter circumstances, the diol epoxides 1 and 2 represented 43 and 38%, respectively, of the total nuclear metabolites. Incubation of liver nuclei with labeled BP or BP-7,8-diol in the presence of NADPH resulted in alkylation of DNA. The alkylated deoxyribonucleosides were separated by Sephadex LH-20 chromatography. Two peaks of radioactivity were noted after incubation with the parent polycyclic hydrocarbon while only one peak was seen after incubation with the diol derivative. These results emphasize the importance of nuclei in the metabolism of BP and in the subsequent alkylation of DNA, reactions which may be related to mutagenesis or carcinogenesis.     

The activity of deoxyribonucleic acid polymerase and deoxyribonucleic acid synthesis in nuclei from brain fractionated by zonal centrifugation

1. The DNA polymerase (EC 2.7.7.7) activity in purified intact brain nuclei from infant rats was investigated. The effects of pH, Mg(2+), glycerol, sonication and storage of the nuclei under different conditions were examined and a suitable assay system was established. 2. The nuclei from infant brain cells were fractionated by zonal centrifugation in a discontinuous sucrose gradient into five zones: zone (I) contained neuronal nuclei (59%) and astrocytic nuclei (41%); zone (II) contained astrocytic nuclei (81%) and neuronal nuclei (19%); zone (III) contained astrocytic nuclei (82%) and oligodendrocytic nuclei (18%); zone (IV) contained oligodendrocytic nuclei (92%) and zone (V) contained oligodendrocytic nuclei (100%). 3. The content of DNA, RNA and protein for each fraction was measured. 4. The distribution of DNA polymerase activity in the fractionated infant and adult rat brain nuclei was determined. The highest activity was found in the neuronal nuclei from zone (I) and the following zones exhibited a progressive decline. In contrast with the nuclei from infant rats those from adults had a much higher activity and expressed a preference for native DNA as template. 5. The deoxyribonuclease activity in all classes of nuclei was measured with [(3)H]DNA as substrate. A general correspondence in the pattern of the relative activities in the nuclear fractions with the distribution of DNA polymerase was found. 6. The incorporation of [(3)H]thymidine into nuclear DNA in infant and adult rat brain was investigated. The specific radioactivity of the DNA in the 10-day-old rats was highest in zone (V) whereas in the nuclei of adult rats, which exhibited a comparatively low incorporation, the highest specific radioactivity was associated with zones (I) and (V).     

Study of nuclear poly(ADP-ribose)polymerase and DNA-topoisomerase II of brain cells during postnatal development of rats

The nuclear poly(ADP-ribose)polymerase activity of neuronal and glial cells during postnatal development of rats was studied. It was shown that the poly(ADP-ribose)polymerase activity of nuclei and nuclear matrix of neuronal cells during postnatal development of rats is increased, whereas the polymerase activity of glial cell nuclei and nuclear matrix in newborn and adult rats is higher than in 14-day-old animals. The DNA-topoisomerase II activity of neuronal nuclear matrix during the postnatal development of rats does not change, whereas the topoisomerase activity of glial nuclear matrix decreases but is always higher than the DNA-topoisomerase II activity of neuronal cell matrix during the postnatal development of rats. It is suggested that ADP-ribosylation in the nuclear matrix of neuronal cells causes the inhibition of the DNA-topoisomerase II activity of nuclear matrix.     

ISOLATION OF CELL NUCLEI FROM THE MAMMALIAN CEREBRAL CORTEX AND THEIR ASSORTMENT ON A MORPHOLOGICAL BASIS

An aqueous method is described for the isolation of highly purified nuclei from the cerebral cortex of adult guinea pigs. Erythrocytes were removed by a short-time perfusion of the brain, myelin fragments by a rapid mechanical method, and blood capillaries by a centrifugal sieving through dense sucrose solutions. The nuclear preparation retained the activity of ATP:NMN adenylyltransferase. Recoveries of DNA in the P4I, P4II, P_L and P_S preparations were 30, 43, 8, and 7%, respectively. Microscopy and phase contrast microscopy showed a satisfactory removal of erythrocytes, myelin fragments, capillaries, and cytoplasmic elements. Biochemical purity of samples was verified by the absence of several cytoplasmic enzyme activities. In the electron microscope, the majority of nuclei showed well-preserved nuclear membranes, with nuclear pores, and were provided with a finely textured nucleoplasm. Occasional contaminants were elements of endoplasmic reticulum and of the endothelium. Assortment of nuclei on a morphological basis showed that 55–65% and 47–53% of nuclei in the P4I and P4II preparations, respectively, consisted of neuronal nuclei. In the P_L preparation, the population of neuronal nuclei ranged between 72 and 83%, while 94–99% of the nuclei in the P_S preparation consisted of smaller nuclei, most likely of oligodendroglial origin.     

RNA synthesis in neuronal and glial cell nuclei from rat cerebral hemispheres during early postnatal development

RNA synthesis in rat cerebral hemispheres at 1, 5, and 10 days of age and the relative contribution brought by neuronal and glial nuclei to RNA synthesis was investigated. The experiments were carried out both in vivo (by i.p. injection of [³H]uridine) and in vitro (either by incubation of tissue slices with [³H]uridine or by determination of RNA polymerase activities). The labeling of RNA decreases from 1 to 10 days of age both in vivo and in vitro; the decrease is of the same extent in neuronal and glial nuclei. RNA polymerase activity Mg²⁺-dependent does not change significantly from 1 to 10 days of age either in total, in neuronal, or in glial nuclei, whereas the Mn²⁺-dependent activity increases significantly over the same developmental period studied. The significance of RNA polymerase assay as an index of in vivo RNA synthesis is discussed.     

Isolation and biochemical characterization of nuclei from chick embryo liver

A procedure is described for the isolation of enzymatically active nuclei from chick embryo liver. It consists of the homogenization of the pooled tissue in 0.32 M sucrose-3 mM MgCl₂ followed by a slow centrifugation. The resulting nuclear pellet is then purified further in a discontinuous density gradient composed of sucrose solutions containing Mg²⁺ ions, the lower portion of the gradient being 2.2 M sucrose-1 mM MgCl₂. Based on DNA recovery, the nuclear fraction isolated by the procedure described contained an average of 62% of the nuclei in the original filtered homogenate. Light and electron microscope examinations showed that 90% of the isolated nuclei were derived from hepatocytes. They appeared intact with well preserved nucleoplasmic and nucleolar components, nuclear envelope, and pores. The isolated nuclei were quite pure, having a very low level of cytoplasmic contamination as indicated by cytoplasmic enzyme marker activities and electron microscope studies. The nuclear fraction consisted of 19.9% DNA, 6.2% RNA, 74% protein, the average RNA/DNA ratio being 0.32. Biosynthetic activities of the two nuclear enzymes NAD-pyrophosphorylase and DNA-dependent RNA polymerase were preserved. The specific activities of these enzymes were: NAD-pyrophosphorylase, 0.049 µmoles nicotinamide adenine dinucleotide (NAD) synthesized/min per mg protein; Mg²⁺ activated RNA polymerase, 4.3 µµmoles UMP-2-C¹⁴ incorporated into RNA/µg DNA per 10 min; and Mn²⁺-(NH₄)₂SO₄ activated RNA-polymerase, 136 µµmoles UMP-2-C¹⁴ incorporated into RNA/µg DNA per 45 min.     

Exchange assays using sodium thiocyanate to measure total nuclear content of estrogen receptors in the hypothalamic pituitary axis of mice

We studied the effect of sodium thiocyanate (NaSCN) in the determination of specific nuclear estrogen receptors from the hypothalamic-pituitary axis (HPA) of mice. We compared the results with those of the exchange assay using either suspensions of whole nuclei or KC1-nuclear extracts. Our findings demonstrated that 0.5 M NaSCN was more efficient than 0.5 M KC1 in extracting [³H] E₂ nuclear content from HPA (91% vs 79.3%). Nuclear fractions extracted with 0.5 M NaSCN revealed the presence of a single class of low-capacity-high affinity binding sites that sedimented in the 4.0 S area. Nuclear binding was T° dependent and reached maximum levels of 450 ± 156 (S.E.) fmol/mg DNA after an overnight incubation at 4°C. Such levels were comparable to those observed in whole nuclei suspensions after 1 hour incubation at 37°C (618 ± 71 fmol/mg DNA, p > 0.05) but two-fold higher (p <0.01) than the concentration of binding sites measured in KC1-extracted nuclear fractions under similar experimental conditions. We conclude that NaSCN extracted the total content of nuclear estrogen receptors in HPA of mature mice.     

RIBOSOMAL RNA PRECURSORS IN NEURONAL AND GLIAL RAT BRAIN NUCLEI

Abstract– The method of T hompson (1973) for isolation and fractionation of brain nuclei was modified by the introduction of 12mM-Mg²⁺ in the isolating media. This technique gives a good yield of pure (85-90%) neuronal and glial rat brain nuclei, with minimal disruption of nuclei and degradation or processing of nuclear RNA. The RNA/DNA ratio of neuronal nuclei is about 3-fold higher than that of glial nuclei. Analysis of nucleolar RNA fractions by urea-agar gel electrophoresis allows the identification of 45S, 41S, 39S, 36S, 32S and 21S pre-rRNA components. The pattern of nucleolar pre-rRNA and rRNA species in neuronal and glial nuclei is identical. These results demonstrate the existence in brain nuclei of multiple pre-rRNA processing pathways qualitatively similar to those observed in other animal tissues.