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細胞性粘菌キイロタマホコリカビにおけるパルスラベルされたRNAを含む核内リボ核タンパク粒子の研究

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(1)Title. 細胞性粘菌キイロタマホコリカビにおけるパルスラベルされたRNAを含む 核内リボ核タンパク粒子の研究. Author(s). 神田, 房行. Citation. 北海道教育大学紀要. 第二部. B, 生物学,地学,農学編, 30(2): 103-143. Issue Date. 1980-03. URL. http://s-ir.sap.hokkyodai.ac.jp/dspace/handle/123456789/6368. Rights. Hokkaido University of Education.

(2) Journal of Hokkaido University of Education (Section II B) Vol. 30, No. 2 March, 1980. C?fti6^t±^i?.3^ (^2 $PB)^ 30 % ^,2^ Bgffi 55 ^ 3 ^. Study on Nuclear Ribonucleoprotein Particles Containing Pulse-Labeled RNA in the Cellular Slime Mold Dictyostelium discoideum. Fusayuki KANDA Biological Laboratory, Kushiro College, Hokkaido University of Education, Kushiro 085. M ft: ^BJ3^?^®^^n^^^3 i^fc'^joit^^^x^^^. ? ^ R NA ^^W )) if^^ ^°^-?^>W£. wm^^^mw^wm Contents. Summary ............................................................ 194. Introduction ........................................................ 105. Materials and Methods .......................................... log (a). Cells. .......................................................... log. (b) Isotopic labeling .......................................... IQS. (c) Cell fractionation---------------------------------- 108 (d) Purification of nuclei .................................109. (e) Extraction of RNP material from nuclei ••••••••• 109. (f) RNA extraction and sucrose gradient centrifugation of RNA ................................. 109. (e) Preparation and use of poly(U)-fiberglass filters. .........................................................no. (h) CsCl density-gradient centrifugation ...........•... m. (i) SDS-polyacrylamide gel electrophoresis ......... in 0) Radioactivity measurement ........................... m (k) Estimation of DNA--------------------------- 112 (1) Isotopes and chemicals ................................. 112 Results ............................................................... 112. (a) Technical reliability for the isolation of nuclei from amoebae ...................•..........•....'•.•.•••.. 112. (103).

(3) Fusayuki KANDA. (b) Sedimentation analysis of nuclear materials containing rapidly labeled RNA ..................... 115. (c) Effect of polyvinylsulfate on rapidly labeled structure ................................................... 117. (d) Effect of Mg2+ on the size of nuclear RNP •••••• 117. (e) CsCl density-gradient analysis of nuclear extracts ...................................................... 120. (f) Characterization of RNA from nuclear RNP particles ................................................... 122. (e) Relationship of the size between nuclear RNA RNA ••••.••••••....••....••••....-•...••.•••••.•••..•.--123. (h) Sedimentation analysis of poly (A) containing RNA ••-•••••••••••••••••••"•••••••••••••••••••••••••••••••• 125. (i) Protein components of nuclear RNP particles in different preparations ................................ 132 Discussion .............................••••••••••••••••••••••••••••••• 136. (a) Extraction efficiency of nuclear RNP particles •• 136 (b) Evidence for the existence of hnRNA-protein complexes ............"............•......•.....•..•.••..... 136. (c) Existence of small-sized RNP particles in D. discoideum •••••••••••••-•-•••••-••••••••••••••••••••••• 137. (d) Heterogeneity of protein components in RNP particles ................................................... 138. (e) Possible role of proteins associated with hnRNA. .........................................................138. Acknowledgements................................................ 140 References ......................................••••••••••••••••••••• 140. Summary Nuclei of the cellular slime mold Dictyostelium discoideum were isolated and purified using a low concentration of the nonionic detergent Triton X-100. The critical concentration of the detergent which could bring about the breakage of almost all of the cells was 0.08% (v/v), and at this concentration the yields of purified and crude nuclei were 69% and 91.3%, respectively. The ribonucleoprotein particles containing rapidly labeled RNA were isolated from the purified nuclei of interphase cells and their properties characterized. The ribonucleoprotein particles were efficiently extracted at pH 8.0. The size of the ribonucleoprotein particles (10S—50S) of this slime mold was considerably small in comparison with that of the nuclear ribonucleoprotein particles found in higher eukaryotes. The small-sized ribonucleoprotein particles were not artificial products due to ribonuclease digestion of large particles. That the rapidly labeled RNA of the. (104).

(4) Nuclear Ribonucleoprotein Particles in D. discoideum. nuclear ribonucleoprotein particles is heterogeneous nuclear RNA was demonstrated by its sedimentation property, the resistance of the synthesis of this RNA species to a relatively high concentration of actinomycin D that selectively inhibits the synthesis of ribosomal RNA, and the existence of polyadenylic acid sequence in the RNA molecules. The size of the ribonucleoprotein particles was proportional to that of the RNA contained in the particles ; the mean size of the RNA obtained from the rapidly sedimenting particles was larger than that of the RNA from the slowly sedimenting ones. The protein content of the ribonucleoprotein particles was estimated to be 38.3% from the result of the CsCl equilibrium densitygradient analysis. The protein components constructing the particles consisted of at least four proteins with the molecular weight of 80,000, 66,000, 60,000 and 42,000 daltons. These proteins were readily distinguishable from the protein components of other cellular organella, including ribosomal proteins or soluble proteins. It was concluded from the experimental results in this study that almost all of the nuclear ribonucleoprotein particles containing rapidly labeled RNA in the interphase amoebae of D. discoideum consist of heterogeneous nuclear RNA and several different proteins in the molecular weight.. Introduction In bacterial cells messenger RNA (mRNA) is known to bind with ribosomes immediately after its synthesis (Stent, 1966). The gene transcription and translation in the bacterial system are coupled with each other ; a transcription-translation complex is composed of DNA molecules as templates, RNA polymerase, newly synthesizing RNA chain and ribosomes translating mRNA (Byrne et al., 1964 ; Shin & Moldave, 1966 ; Jones et al., 1968 ; Martin, 1969). In eukaryotes, on the other hand, the two phenomena of the gene transcription and translation are separated in space by the nuclear membrane ; the transcription takes place in the nucleus,. while protein synthesis is localized in the cytoplasm (Muramatsu, 1971 ; Darnell et at., 1973). The transport of RNA from the nucleus to the cytoplasm will become an important process for the expression of genes.. When mammalian cells were labeled for a short period with a radioactive RNA precursor, most of the radioactivity was found in the heterogeneous RNA fraction in the nucleus. This kind of the RNA species is termed a heterogeneous nuclear RNA (hnRNA) by a number of authors (Warner et al., 1966 ; Penman et al., 1968 ; Darnell et al., 1973 ; Kolata, 1974). In rat liver or Ehrlich ascites carcinoma cells, the hnRNA comprised about one third of the total nuclear RNA (Georgiev & Samarina, 1971). The hnRNA possesses several properties typical of mRNA as indicated below (Georgiev & Mantieva, 1962 ; Jacob & Bush, 1967 ; Muramatsu, 1971) ; (1) DNA-like base composition, (2) a high ability in the molecular hybridization with DNA, (3) an ability to stimulate amino acid incorporation into proteins in a cell free system, (4) a heterogeneous distribution in the sucrose gradient analysis and (5) a continuation of the synthesis at the concentration of actinomycin D that selectively inhibits the synthesis of ribosomal RNA (Roberts & Newman, 1966 ; Penman et al., 1968 ; Mizukami & Iwabuchi, 1970b ; Firtel et al., 1973). For. (105).

(5) Fusayuki KANDA. several reasons including the fact that about 30% of these molecules contain a polyadenylic acid (poly(A)) sequence at the 3'-end, it is thought that at least a part of the hnRNA molecules serves as precursors to mRNA (Darnell, 1968 ; Darnell etal., 1971, 1973 ; Philipson etal., 1971 ; Greenberg & Perry, 1972 ; Jelinek et al., 1973, 1974 ; Brawerman, 1974). It has been shown that newly formed hnRNA has a very high molecular weight. The distribution peak of newly formed hnRNA in the sucrose gradient is observed in the zone corresponding to the molecular weight of 2—20X106 daltons. Thus, the molecular weight of the hnRNA synthesized in the cell nucleus is much higher than that calculated for monocistronic mRNA (Yoshikawa-Fukada etal., 1965 ; Attardi et al., 1966; Scherrer et al., 1966 ; Granboulan & Scherrer, 1969). Since the molecular weight of cytoplasmic mRNA is lower than that of hnRNA and roughly corresponds to that of monocistronic mRNA, the bulk of hnRNA sequences is rapidly degraded in the nucleus and as a result only a relatively small fraction of the rapidly labeled RNA is conserved, reaching the cytoplasm as mRNA (Attardi et al., 1966 ; Penman et al., 1966, 1968 ; Scherrer et al., 1966 ; Shearer & McCarthy, 1967 ; Soeiro et al:, 1968). The function of the hnRNA which degrades within the nuclei has not been clear as yet, although several workers have proposed some hypotheses on its function (Frenster, 1965 ; Scherrer et al., 1966 ; Britten & Davidson, 1969). The large-sized hnRNA in higher eukaryotes has been found to be complexed with protein. In 1965, Samarina et at. reported in animal cells that hnRNA exists as the nucleoprotein complex in the nuclei but not as a free form (Samarina et al., 1966). Later the nucleoprotein complex was also found in other mammalian cells (Lawford etal., 1967 ; Georgiev, 1968, 1972 ; Kohler & Arends, 1968 ; Moule & Chauveau, 1968 ; Parsons & McCarthy, 1968 ; Ishikawa et al., 1969, 1972, 1974 ; Faiferman et al., 1970 ; Martin & McCarthy, 1972 ; Cornudella et at., 1973 ; Sommerville, 1973 ; Pederson, 1974a ; Quinlan et al., 1974 ; Kish & Pederson, 1975 ; Kumar & Pederson, 1975 ; Naora, 1975 ; Raj et at., 1975). This complex bears almost all of nuclear RNA exhibiting DNA-like base composition and no substances besides RNA and protein (Samarina et at., 1967a, 1967b ; Lukanidin et al., 1971, 1972b). If the nucleoprotein complexes are isolated in the presence of RNase inhibitors, they have very high sedimentation coefficients of 503 to 300S (Samarina et at., 1968). In the absence of the inhibitor, these large complexes are degraded to a homogeneous population of monomer particles with a sedimentation coefficient of about SOS (Samarina et al., 1968). The structures resembling the above-mentioned complexes have been seen with the electronmicroscope in the section of nuclei (Georgiev & Samarina, 1971). In the case of mRNA as well as hnRNA, it was reported that there were mRNA complexes with protein. Spirin et al. (1965) reported in a sea urchin embryo that rapidly labeled cytoplasmic RNA with the characteristic properties of mRNA complexes with protein. These RNP particles, which are free cytoplasmic mRNA-protein complexes not associated with ribosomes, were named informosomes (Spirin & Nemer, 1965 ; Spirin, 1966, 1969 ; Yamana & Shiokawa, 1967 ; Infante & Nemer, 1968 ; Kafatos, 1968 ; Mano, 1968 ; Spohr et al., 1970, 1972 ; Sarkar & Moscona, 1971 ; Stepanow etal., 1971; Woodcock & Mansbridge, 1971; Jacobs-Lorena & Baglioni, 1972 ; Dubochet et al., 1973 ; Gross et al., 1973 ; Ruzdijic et al., 1973 ; Bag & Sarkar, 1975). Another type of cytoplasmic ribonucleoprotein materials containing mRNA has been obtained from cytoplasmic. (106).

(6) Nuclear Ribonucleoprotein Particles in D. discoideum. polysomes (Cartouzou et al., 1968 ; Henshaw, 1968 ; Perry & Kelley, 1968 ; Samec et al., 1968 ; Temmerman & Lebleu, 1969 ; Leytin etal., 1970 ; Lissitzky etal., 1970 ; Lowenstein, 1970 ;Spohr etal., 1970, 1972 ; Lebleu etal., 1971; Lee & Brawerman, 1971; Lee etal., 1971 ; Blobel, 1972, 1973 ; Kumar & Lindberg, 1972 ; Kwan & Brawerman, 1972 ; Bryan & Hayashi, 1973 ; Lindberg & Sundquist, 1974 ; Harrison etal., 1974 ; Sommerville, 1974 ; Auerbach & Pederson, 1975 ; Chen et al., 1976). In contrast to mammalian cells, some of lower eukaryotes do not have large-sized hnRNA.. In a unicellular eukaryote, Tetrahymena puriformis, Kumar (1970) has reported that the RNA with the S value greater than 35S was not detected in the nuclei. With the same organism, Prescott et al. observed the heterodisperse RNA sedimenting in the region from 4S to SOS. It was also reported that Phycomyces blakesleeanus, multinucleated fungs, and Amoeba proteus do not have high molecular weight hnRNA (Gamow & Prescott, 1972). In yeast, the size of hnRNA containing poly(A) is shown to be about US, slightly higher than that of cytoplasmic mRNA (Shiokawa & Pogo, 1974). In the cellular slime mold, Dictyostelium discoideum, which belongs to a typical lower eukaryote and has become popular as an experimental system to study the biochemical process in-. volved in cell differentiation, a lot of morphological and biochemical knowledge has been accumulated (Mizukami & Iwabuchi, 1970a ; Firtel & Brackenbury, 1972 ; Lodish et al., 1974 ; Tuchman et al., 1974 ; Yagura et al., 1976 ; Yanagisawa et al., 1977 ; Kanda, 1979a). The cellular slime mold offers certain advantages over higher eukaryotes for the analysis of differentiation particularly at the level of gene transcription and translation. During its life cycle, this microorganism displays qualitative changes in RNA transcript (Firtel, 1972) as well as changes in the specific activities of various enzymes (Loomis, 1970 ; Telser & Sussman, 1971). The changes appear, at least in part, to be regulated at the level of transcription (Firtel et al., 1973). In particular, Firtel et al. reported that this organism has no large-sized hnRNA and the sedimentation coefficient of hnRNA is about 15S (Firtel et al., 1973 ; Firtel & Lodish, 1973). The hnRNA is shown to be only 20% larger on the average than cytoplasmic mRNA and contains at least one sequence of poly(A) (Firtel etal., 1972 ; Firtel & Lodish, 1973 ; Kanda, 1975, 1977a, 1977b,1977c). In D. discoideum, in contrast to the situation of higher organisms, no significant proportion of newly formed RNA appears to turn over inside the nucleus and almost all of the nuclear RNA is probably transported to the cytoplasm (Kessin, 1973 ; Soil & Sussman, 1973 ; Jacobson et al., 1974). The above evidence seems to suggest that in the lower eukaryotes the process of the trans-. port of the genetic message of DNA to the cytoplasm is simpler as compared with that in mammalian cells. Thus, it is now interesting to know the manner of existence of the small-sized. hnRNA in D. discoideum. If the similarity in the size of the hnRNA and mRNA in this organism displays a simplified system of the transport of the genetic message, the hnRNA may exist as a free form, or the protein content of the postulated RNP may be lower than that reported for the mammalian RNP.. The present study was designated to search the manner of existence of hnRNA in D. discoideum from the viewpoint of the control of the transport of the genetic message from the nucleus to the cytolasm. In this paper, it is described that D. discoideum possesses nuclear RNP. (107).

(7) Fusayuki KANDA. particles of which the average size is small relatively to that observed in higher eukaryotes, and the protein component of the RNP particle of this organism is heterogeneous as compared with that of the mammalian RNP particle.. Materials and Methods. (a) Cells The amoeba cells of D. discoideum, strain NC-4 (haploid), were grown in liquid medium containing autoclaved Escherichia coli cells (Banner, 1947 ; Ito & Iwabuchi, 1970, 1971 ; Iwabuchi et al., 1970 ; Mizukami & Iwabuchi, 1972 ; Samesima etal., 1972). When the cell density reached about 3 X 107 /ml in the late log-phase of growth, the cells were harvested, washed several times by centrifugation and suspended in a TKM buffer (15 mM Tris-HCl (pH 7.6), 20 mM KC1 and 5 mM MgCy containing streptomycin sulfate (150 ^cg/ml) to avoid contamination with living bacteria at a cell density of 3 X 10 /ml. In order to obtain a high degree of synchrony of morphogenesis, definite volumes of the cell suspension were dispensed on non-nutrient agar plates containing streptomycin (150 //g/ml), followed by incubation at 23°C in the dark under sterile conditions. The cells which proceeded to the early aggregation stage of morphogenesis (5 to 6 hr after the onset of incubation) were used throughout the present study.. (b) Isotopic labeling The cells in the early aggregation stage of morphogenesis were harvested and washed three times by centrifugation with a TKM buffer and suspended in the same buffer at a cell density of 109/ml. The cells were incubated with 120 /^g/ml of actinomycin D for 1 hr and then exposed to 500 /^Ci/ml of [3H]uracil for 30 min in the presence of the drug. After labeling, the cells were rapidly chilled and washed with an ice-cold TKM buffer by centrifugation. (c) Cell fractionation All the procedures for the preparation of nuclei and cytoplasmic fractions were conducted at 0°-4°C.. The labeled cells were washed several times by centrifugation with an ice-cold TKM buffer. In the final washing, the cells were pelleted by centrifugation at 5,000 Xg for 10 min at 4°C. After centrifugation, the cells were resuspended in a 5% sucrose solution containing Triton X-100, 3 mM magnesium acetate and 10 ^g/ml polyvinylsulfate (PVS) at a concentration of 2X 108 cells/ ml. The cells were then disrupted in a loose-fitting Potter-type homogenizer. After standing for 10 min, an equal volume of 22% sucrose containing 3 mM magnesium acetate and 10 p.g/m\ PVS were added to the cell lystate. It was then centrifuged at 3,000 rev./min for 10 min in an RPR 18 rotor of a Hitachi centrifuge. The pellet was washed further twice by centrifugation with 13.5% sucrose containing 0.1% Triton X-100, 3 mM magnesium acetate and 10 //g/ml PVS, and the finally obtained pellet was named "crude nuclear fraction .The supernatant fraction was. centrifuged at 10,000rev./min for 10 min in a ft 1 rotor of a Kubota centrifuge. The supernatant. (108).

(8) Nuclear Ribonucleoprotein Particles in D. discoideum. fluid obtained in this centrifugation was named "soluble cytoplasmic fraction" and the sedimented material named "mitochondrial fraction".. (d) Purification of nuclei The purification of nuclei was conducted by a slight modification of the method of Cocucci and Sussman (1970) (Kanda, 1979b). The crude nuclear pellet was resuspended in 4 ml of 0.25 M sucrose containing 3 mM magnesium acetate and 10 ^g/ml P VS followed by the addition of 8 ml of 2.3 M sucrose containing 3 mM magnesium acetate and 10 /^g/ml PVS. The suspension containing crude nuclei was layered over 15 ml of 1.95 M sucrose solution containing 3 mM magnesium acetate and 10 ^g/ml PVS, and then centrifuged for 45 min at 17,000 rpm and 4°C in an SW 25.1 rotor of a Spinco L ultracentrifuge. The nuclei collected through 1.95 M sucrose solution were termed purified nuclei". The microscopic observation of the purified nudear preparation revealed that the nuclear sample contained unbroken cells less than 0.1% of the total cells but no significant amount of cytoplasmic material. (e) Extraction of RNP material from nuclei The purified nuclei were suspended in a TNM buffer (10 mM Tris-HCl, 100 mM NaCl and 1 mM magnesium acetate (pH 7.0)) containing 10 ^g/ml PVS, stirred for 10 min at 4°C and centrifuged at 7,000 X g for 10 min. The resultant supernatant fluid was termed "pH 7.0 nuclear extract". The residual pellet was resuspended in a TNM buffer (pH 8.0) containing 10 ^/g/ml PVS, stirred for 30 min at 4°C and centrifuged at 10,000 Xg for 10 min. The supernatant fluid so obtained was named "pH 8.0 nuclear extract". The residue was finally suspended in a TNM. buffer (pH 8.0) containing 10 //g/ml PVS, followed by two cycles of the freezing-thawing procedure (freezing in an ethanol-dry ice bath (—70°C) and thawing at 4°C) and then centrifuged at 10,000 Xg for 10 min to remove nucleoli and nuclear debris. The supernatant fluid so obtained was named freezing-thawing nuclear extract". All operations were carried out at 0°—4°C.. Usually 1.5—2.0 ml of the nuclear extract were layered onto the top of a 10—25% (w/v) linear sucrose gradient (32 ml) containing 2M sucrose cushion at the bottom which was made with TNM (pH 8.0) containing 10 ^g/ml PVS and then centrifuged at 26,000 rev./min and 4°C for 14 hr using a Hitachi RPS 27 rotor, (f) RNA extraction and sucrose gradient centrifugation of RNA The extraction of RNA from RNP material was conducted by the sodium dodecylsulfate (SDS)-phenol procedure, as reported by Iwabuchi et al. (Iwabuchi et al., 1970, 1971 ; Mizukami & Iwabuchi, 1970b). For the preparation of rapidly labeled RNA from the RNP particles, the nuclear extracts were fractionated by means of sucrose gradient centrifugation. Then, RNP particles. were pooled after the measurement of radioactivity and diluted with 10 mM Tris-HCl (pH 7.6)-0.1 M NaCl-20 //g/ml PVS. To the solution containing the RNP particles or total nuclear extract was added 10% SDS at a final concentration of 1% and an equal volume of phenol- m -cresol-water (7 : 1 : 2, in volume) containing 0.1% 8-hydroxyquinoline. The mixture was stirred for 20 min in the cold and centrifuged to obtain an aqueous fraction. The extraction was repeated twice. All. (109).

(9) Fusayuki KANDA. the aqueous fractions were combined and repeatedly deproteinized by treatment with the phenol-m -cresol-water mixture. The RNA in the final extract was precipitated by the addition of 2.5 volumes of 95% ethanol at —30°C after treatment with ether. All operations were carried out at 2°-4°C.. In the sucrose gradient analysis of RNA prepared from the nuclear extract or isolated RNP, the RNA sample was dissolved, unless otherwise stated, in a buffer A (20 mM Tris-HCl (pH 7.6), 0.1 M NaCl, 2 mM sodium ethylenediaminetetraacetic acid (EDTA) and 20 /^g/ml PVS), mixed with unlabeled 26S and 17S rRNA's of D. discoideum as internal reference and centrifuged at 26,000 rev./min and 8°C for 15 hr using a Hitachi RPS 27 rotor. After centrifugation, the gradient was fractionated, 2 ml of water were added to each fraction and the absorbance at 260 nm was measured.. Poly(A)-containing RNA was prepared by the method of Firtel et al. (^irtel et al., 1972 ; Firtel & Lodish, 1973 ; Perry & Kelley, 1973 ; Perry et at., 1972, 1974). The soluble cytoplasmic extract was mixed with 15 ml of buffer B (25 mM Tris, 5 mM EDTA and 2.6% SDS, pH 7.6), treated with 1% diethylpyrocarbonate for several seconds, and RNA was extracted with a solution of phenolchloroform-isoamylalcohol (49 : 49 : 2). During the third extraction, 0.1 volume of 4 M sodium acetate (pH 5) was added. The RNA was precipitated with 2.5 volumes of cold 95% ethanol. For the preparation of poly(A)-containing RNA from crude nuclei, they were suspended in 10 ml of buffer B, and the RNTA was extracted according to the procedure used for the cytoplasmic RNA. For the sucrose gradient analysis of poly(A)-containing RNA, the RNA sample was dissolved in a buffer C (10 mM Tris-HCl (pH 7.6), 0.1 M NaCl and 0.05% SDS), mixed with unlabeled rRNA's of D. discoideum and centrifuged in 4.8 ml of a 15—30% (w/v) linear sucrose gradient in the buffer C for 2 hr at 48,000 rev./min and 22°C in a Spinco SW 50 rotor. After centrifugation, an appropriate number of fractions of the gradient were taken. To each fraction was added 2 ml of 10 mM Tris-HCl, 0.12 M NaCl, 0.1 mM EDTA and 0.5% SDS, pH 7.6. These fractions were then filtered through polyuridylic acid (poly(U))-fiberglass filters. (e) Preparation and use of poly(U)-fiberglass filters In the examination of poly(A)-containing RNA, the poly(U)-filter method was adopted for the RNA fractionated by sucrose gradient centrifugation. The poly(U)-fiberglass filters were prepared according to the method developed by Sheldon et al. (1972). To a fiberglass filter (Whatman GF/C, 2.4 cm in diameter) supported on a rubber grid was added 0.15 ml of poly(U)-solution (1 mg/ml in distilled water). The filters were dried at 37°C for 2 hr and then irradiated for several minutes with ultra-violet light on each side at a distance of 22 cm from a 10 W germicidal lamp. Each filter was rinsed with about 50 ml of distilled water to remove any unbound poly(U). The isotopically labeled RNA which was extracted from the soluble cytoplasmic fraction prepared from cells labeled with 3H-uracil for 30 min in the presence of actinomycin D (120 p. g/ ml) was tested for binding to poly(U). The RNA was dissolved in the binding buffer (10 mM Tris-HCl, 0.12 M NaCl, 0.1 mM EDTA and 0.5% SDS, pH 7.6) and then filtered at the flow rate of. (110).

(10) Nuclear Ribonucleoprotein Particles in D. discoideum. 2 ml/min through poly(U)-fiberglass filters. The crude nuclear RNA containing poly(A) was centrifuged on sucrose gradients and then : ractionated. A half portion of each fraction was used for the measurement of the absorbance at 260 nm and the radioactivity. The remaining portion was adjusted to 10 mM of Tris-HCl (pH 7.6), 0.12 M of NaCl, 0.1 mM of EDTA and 0.5% SDS at the final concentration and then passed through poly(U)-filters. The filters were washed with 20 ml of the binding buffer, followed by 10 ml of 0.3 M ammonium acetate in 50% ethanol. They were dried and the radioactivity was measured (Firtel et al., 1972). (h) CsCl density-gradient centrifugation The buoyant density analysis of RNP particles was carried out as detailed by Mizukami & Iwabuchi (1972). The RNP particles or nuclear extracts were fixed by the addition of 40% glutalaldehyde (Fisher Scientific Co., Biological grade) which was neutralized to pH 7.0 with 1 M NaHCOa to a final concentration of 6% at 4°C (Spirin etal., 1965 ; Perry & Kelley, 1966 ; Baltimore & Huang, 1968). A 0.2—0.5 ml portion of the fixed sample was layered on the top a preformed CsCl density-gradient in a TNM buffer and centrifuged at 37,000 rev./min for 17 hr at 4°—6°C in the Spinco SW 50 rotor. 0.15 ml fractions were collected from the bottom of the tubes, and the density of every fourth fraction was determined from the index according to the formula pzo= 10.860—13.492 g/cm3 (Meselson etal., 1964). Each fraction was then mixed with 1.0 ml of water, and 50 //g of yeast RNA as carrier and 1.5 ml of 20% trichloroacetic acid-10% acetone were added.. (i) SDS-polyacrylamide gel electrophoresis After the sucrose gradient centrifugation of nuclear extracts, the fractions containing the RNP structure were pooled and then 0.1 volume of cold 100% (w/v) trichloroacetic acid was added to them (Kumar & Warner, 1972). The precipitated material was collected by centrifugation at 12,000 Xg for 20 min at 0°C, dried, and dissolved in 0.1 ml of 0.1 M sodium phoshate (pH 7.0) containing 1% SDS, 1% 2-mercaptoethanol and 4 IV[ urea (Kanda et al., 1974). It was incubated for 3 hr at 37°C and then electrophoresed on 10% polyacrylamide gels containing 1% SDS for 12 hr at a constant current of 5 mA/gel (Weber & Osborn, 1969 ; Ochiai etal., 1971, 1973 ; Kanda et al., 1972, 1974). For the determination of the molecular weight of proteins, several marker proteins were parallely run in separate gels (Kanda, 1978). After electrophoresis, the gels were stained with 0.2% coomassie blue in 7% acetic acid-10% ethanol and destained in the above acetic acid-ethanol mixture. The destained gels were scanned at 570 nm with a 0.5-mm wide beam in the scanning attachment of the Ozmor recording spectrometer, type 82L. The absorbance was recorded at a scanning speed of 2 cm/min and a chart speed of 4 cm/min. Peak areas. in the absorbance profile were represented by the integral value of absorbance. 0)' Radioactivity measurement The isotopically labeled RNA and RNP were precipitated in 10% trichloroacetic acid containing 5% acetone and collected on a fiberglass filter (Whatman GF/C). The filters were. (Ill).

(11) Fusayuki KANDA. washed with 5% trichloroacetic acid, dried and counted in a Beckman liquid scintillation spectrometer (Iwabuchi et al., 1971). The radioactivity of the samples doubly labeled with 3H- and 14C-compounds were counted with the aid of the double channel setting of 3H and 14C (Iwabuchi et al., 1971).. (k) Estimation of DNA The amount of DNA was estimated according to the method advanced by Burton (1956) (see Dische, 1930). Five ml of difenylamine solution were added to 2.5 ml of the sample solution containing DNA. The mixture stood for 16—20 hr at 30°C and ODeoonm was then measured. The. diphenylamine solution was prepared as follows. Dephenylamine (1.5 g) was dissolved in 100 ml of glacial acetic acid and 1.5 ml of H2S04 was added. Before use, 0.5 ml of acetoaldehyde solution (0.2 ml acetoaldehyde in 9.8 ml of distilled water) was further added to the dye solution. (1) Isotopes and chemicals 3H-uracil (53 Ci/mmole) was obtained from Schwartz BioResearch Inc., Orangeburg, N. Y.,. and 14C-labeled Chlorella protein hydrolysate (7.1 mCi/mmole) was a gift from the Institute of Applied Microbiology, University of Tokyo, through the Japan Isotope Association. Actinomycin D was purchased from Mann Research Labs., (New York, N. Y.). Poly(U) was obtained from Sigma Chemical Co.. Results (a) Technical reliability for the isolation of nuclei from amoebae For the isolation of the nuclei of D. discoideum amoeba cells, Cocucci and Sussman (1970) have used Cemulsol NPT 12 as the detergent to lyze plasma membrane. Their method was developed by some workers and has been commonly used for the isolation of nuclei in the investigation of nuclear DNA (Firtel & Banner, 1972 ; Firtel, 1972) and RNA (Cocucci & Sussman, 1970 ; Firtel et al., 1972 ; Firtel & Lodish, 1973). However, the nature of Cemulsol NPT 12 has not been clarified as yet and the concentrations used in the experiments were always much higher than those of other deter gents used for the preparation of nuclear RNP particles of mammalian cells (Perry & Kelley, 1968). A Triton X-100 of a non-ionic detergent, has been usually employed as a useful tool for the isolation of mammalian nuclei. Perry and Kelley (1968) used Triton X-100 to break the plasma membrane of cultured L-cells as described below. The L-cells were suspended in 8 volumes of 10 mM Tris-HCl buffer (pH 7.8) containing 1 mM MgCl2, 0.3 M sucrose and 0.05% Triton X-100 and homogenized with a Bounce homogenizer. This method could not make any damage to the nuclei so far as the nuclear preparation was microscopically observed (Perry & Kelley, 1968). Recently Aaronson & Blobel (1974) have showed that the nuclear membrane of a rat liver can be completely removed by high concentrations of Triton X-100 (2%) but the membrane-deduced nuclei do not show any change in either the shape or the internal ultrastructure. Since the. (112).

(12) Nuclear Ribonucleoprotein Particles in D. discoideum. detergent treatment is able to solubilize approximately 10% of the total nuclear protein, the proteins obtained thus seem to probably be few specific classes of the major proteins existing in the nuclear membrane. Moreover, when the crude nuclei are washed in a low concentration of. Triton X-100, the ribosomes attached to the outeriayer of the nuclear membrane are freed (Ueda, 1972). It has been also shown that polysomes isolated by using Triton X-100 support the incorporation of amino acids in a cell-free system, but this detergent does not remove any protein. that semms to bind to the mRNA in polysomes (Olsnes, 1970, 1971). From the above reasons, the author has used Triton X-100 as a detergent for the disruption of the plasma membrane and the preparation of the purified nuclei of the cellular slime mold (Kanda,. 1979b). The amoeba cells of D. discoideum were harvested with an ice-cold TKM buffer (see Materials and Methods) and washed several times by centrifugation. The cells were then suspended in the solution containing Triton X-100 and homogenized in a loose-fitting Potter-type homogenizer with several strokes. The cell disruption was microscopically monitored. The cell lysate was centrifuged to collect intact nuclei. Initially, Brij 58 of a non-ionic detergent was used to disrupt the plasma membrane of D. discoideum. However, this detergent exerted no effect on the disruption of the plasma mem-. brane of the'amoeba cells of this organism. Although the concentration of this agent was increased to 1%, only 16% of the initial cells were broken. On the other hand, when Triton X-100 was adopted for the cell breakage, cells lysed rapidly and a large number of nuclei were obtained which appeared intact under microscopy. The critical concentration of Triton X-100 which could bring about the breakage of almost all of the cells was 0.08% (v/v). Thus, D. discoideum amoebae were routinely suspended in the solution containing 0.08% Triton X-100 at the cell density of 2X10" cells/ml and homogenized with several strokes in the Potter-type homogenizer. Four to five % of the total cells remained unbroken after homogenization. Then the suspension of homogenized cells stood for 10 min, about one half of the remaining-unbroken cells was disrupted. The rest portion (1 to 2%) of the unbroken cells could be further disrupted during the washing of the crude nuclei with the solution containing 0.1% Triton X-100. Table I shows the effect of magnesium ions on the stability of nuclei. The stability of nuclei was measured by the amount of DNA which remained in the crude nuclear fraction plus the mitochondrial fraction. The amoeba cells were divided into two parts ; one of which being suspended in 0.08% Triton X-100 solution containing 1 mM magnesium acetate and the other in 0.08% Triton X-100 solution containing 10 mM magnesium acetate at the cell density of 2x10 cells/ml. As shown in Table I, there is no significant difference in the amount of DNA in the crude nuclear plus mitochondrial fractions between two different Mg2+ concentrations used in the preparation of crude nuclei. However, in the above cases the nuclear preparation were con-. taminated with the cytoplasmic material. The above result suggests that at 1 mM Mg + a small number of nuclei were broken during the course of the purification of nuclei so that the nuclear DNA was solubilized in the cytoplasmic fraction. Thus, in the subsequent experiments, the Mg2+ concentration used for the preparation of nuclei was increased to 3 mM.. (113).

(13) Fusayuki KANDA. Table I. Effect of Mg2+ concentration on the stability of nuclei. Concentration prar-i-inn OD_ ^ ins ofMg2+-~""- rracuon ""600 x luo '70. nuc!eL_+..:_ 274.5 98.7 mitochondria ^>-r.^. soluble cytoplasm 3.5 1.3. lOmM. nucieL.+^ 281.0 100.0 mitochondria ^"x.u xuu.. soluble cytoplasm 0.0 0.0. The amoeba cells of D. discoideum which proceeded to the early aggregation stage of morphogenesis were harvested and washed by centrifugation with ice-cold TKM buffer. In the last washing, the cells were collected at 5,000 Xg for 10 min at 4°C. After centrifugation the cells were suspended in the 5% sucrose solution containing 0.08% Triton X-100, 1 mM or 10 mM magnesium acetate at a concentration of 2X108 cells/ml. The cells were then disrupted in a loose-fitting Potter-type homogenizer. After standing for 10 min, an equal volume of 22% sucrose containing 1 mM magnesium acetate was added to the cell lysate. It was then centrifuged at 3,000 rev./min for 10 min in an RPR 18 rotor of a Hitachi centrifuge. The pellet was further twice washed by centrifugation with 13.5% sucrose containing 0.1% Triton X-100 and 1 mM or 10 mM magnesium acetate. The washed pellet was named the "crude nuclear fraction". The resultant supernatant was further centuifuged at 10,000 .rev./min for 10 min in a # 1 rotor of a Kubota centrifuge. The supernatant fluid and the pellet were named the "soluble cytoplasmic fraction" and "mitochondrial fraction", respectively. The amount of DNA was estimated according to the method advanced by Burton (1956).. Table II shows the yield of nuclei at several steps in the purification process of nuclei. The yield of nuclei was determined by the amount of DNA in each step. The yield of purified nuclei was 69% at the final step. If only the crude nuclei are required, the yield of nuclei could be increased to 91.3%.. In the following experiment, 0.2% Triton X-100 was routinely used to achieve a complete breakdown of the plasma membrane, so that the final procedure for the preparation of crude nuclei was as follows ; the amoeba cells were suspended in 5% sucrose solution containing 0.2% Triton X-100 and 3 mM magnesium acetate and then homogenized with 10 strokes in the Potter-type homogenizer. After standing the cell homogenate for 10 min, an equal volume of 22% sucrose solution containing 3 mM magesium acetate (without Triton X-100) was added. The diluted homogenate was centrifuged for 10 min at l,OOOXg and 4°C. The pellet obtained was washed twice by centrifugation at l,OOOXg for 10 min with 13.5% sucrose solution containing 0.1% Triton X-100 and 3 mM magnesium acetate.. In the process of the preparation of nuclei, the Tris-HCl buffer has not been employed, because it may solubilize the RNP particles in the nuclei so that the particles may be lost during the preparation of nuclei (Muramatsu, 1972).. (114).

(14) Nuclear Ribonucleoprotein Particles in D. discoideum. Tabl II. Yields in various steps during the preparation of nuclei Fraction ODgOo x 103 % Total cell 207 100.0 Crude nuclei 189 91.3 Purified nuclei 143 69.1 The cells were divided into three equal portions. Two-thirds of the cells were suspended in a 5% sucrose solution containing 0.08% Triton X-100, 3 mM magnesium acetate at a concentration of 3Xl08 cells/ml. The cells were then disrupted in a Potter-type homogenizer. After standing for 10 mim, an equal volume of 22% sucrose containing 3 mM. magnesium acetate was added to the cell lysate. It was then centrifuged at 3,000 rev./min for 10 min in an RPS 18 rotor of a Hitachi centrifuge. The pellet fraction was further twice washed by centrifugation with 13.5% sucrose containing 0.1% Triton X-100 and 3 mM magnesium acetate. The washed pellet was named crude nuclei. A-half portion of the crude nuclei was resuspended in 4 ml of 0.25 M sucrose containing 3 mM magnesium acetate, followed by the addition of 8 ml of 2.3 M sucrose containing 3 mM magnesium acetate. The suspension containing crude nuclei was layered over 15 ml of 1.95 M sucrose solution containing 3 mM magnesium acetate, and then centrifuged for 45 min at 17,000 rev./min and 4°C in an SW 25.1 rotor of a Spinco L ultracentrifuge, The nuclei collected through the 1.95 M sucrose solution were termed purified nuclei. The amount of DNA in whole cells, crude nuclei and purified nuclei was estimated as detailed in "Materials and Methods".. (b) Sedimentation analysis of nuclear materials containing rapidly labeled RNA The amoeba cells of D. discoideum were pulsely labeled with 3H-uracil in the early aggregation stage of morphogenesis, following a pretreatment with actinomycin D (120 ^/g/ml) for 1 hr. After labeling, three kinds of nuclear extracts were prepared from purified nuclei under different extraction conditions (at pH 7.0 and 8.0 using freezing-thawing methods). The radioactivity of 3H-uracil incorporated into the acid-insoluble material of these nuclear extracts is shown in Table III. When the nuclear extract was prepared mildly at pH 8.0 or drastically by the freezing-thawing method, approximately 34% or 21% of total 3H-labeled materials present in nuclei were recovered, respectively, whereas the extraction efficiency for the. labeled material obtained with the buffered solution of pH 7.0 was very low (about 5%). Consequently, the technique with the pH 8.0 buffer was the most effective in these three methods. used for the extraction of nuclear labeled materials. Figure 1 shows the sedimentation profiles after the sucrose gradient centrifugation of the nuclear extracts prepared under three different conditions from cells labelled pulsely with 3Huracil. Labeled materials were sedimented heterodispersedly in the region ranging from 10S to SOS with a broad peak in the 20S region. The sum of the radioactivity of the labeled material sedimenting in the region from 10S to 508 was approximately 88% and 80% of the total radioactivity in the pH 7.0 and 8.0 nuclear extracts, respectively. In addition, the radioactivity was scarcely observed in the slowly sedimenting material. In the case of a nuclear extract by the. (115).

(15) Fusayuki KANDA. Table III. Extraction efficiency of rapidly labeled materials from D. discoideum nuclei under three different conditions. Fraction. Radioactivity. pH 7.0 extract. 2,800. % 4.91. pH 8.0 extract. 19,500. 34.22. Freezing-thawing extract. 11,720. 20.57. Residual precipitate. 22,960. 40.29. Nuclei were isolated and purified from D. discoideum amoebae that had been labeled for 30 min with 3H-uracil in the presence of 120 //g/ml actinomycim D. The nuclear material was extracted with TNM buffer of pH 7.0 and pH 8.0 and by the freezing-thawing method as detailed in "Materials and Methods". The radioactivity of 3H-uracil incorporated into the acid-insoluble material was then determined.. ie. pH8D b. 305. v^. ,e x. 16 .c. -» c 3. T< 6L. 2. I II "0~. bottom. ^\Q 2T. Fraction No.. top. -1020" 'Fraction" No. top. Fig. 1 Sedimentation analysis of the nuclear extracts prepared with TNIVt. buffer at pH 7.0 (a) and pH 8.0 (b) and by the freezing-thawing method (c), after labeling of cells with [3H]uracil for 30 min in the presence of the 120 jug/mi actinomycin D. The nuclear extracts were obtained as described in "Materials and Methods". The nuclear extracts were mixed with a trace amount of l4C-labeled SOS ribosomal subunits of E. coli Q13 as a reference to determine the. sedimentation coefficient and centrifuged using a Hitachi RPS 27 rotor at 26,000 rev./min for 14 hr at 4°C in a 10-25% (w/v) linear sucrose gradient containing 2 M sucrose cushion in the bottom. which was made with TNM (pH 8.0) containing 10 /u g/ml PVS.. a, pH 7.0 nuclear extract ; b, pH 8.0 nuclear extract ; c, freezingthawing nuclear extract.. (116).

(16) Nuclear Ribonucleoprotein Particles in D. discoideum. freezing-thawing method, the sum of the radioactivity in the 10—508 region comprised about 46.3% of the total counts and almost all of the counts (about 52.5%) in the rest were found in the region lighter than 10S. However, in the case of the analysis of the pH 7.0 or 8.0 nuclear extract, the radioactivity was scarcely found in the slowly sedimenting material. From the above results, it seems apparent that labeled materials sedimenting faster than 503 were hardly present in these three kinds of nuclear extracts.. To know whether the 10—508 materials containing rapidly labeled RNA are partial degradation products due to the ribonuclease digestion of certain nuclear structures, which occur during the process of the isolation of nuclei or during the preparation of nuclear extracts, or if they are the intrinsical entities in this organism, RNA was separately extracted from the crude nuclei and the nuclear extracts prepared under the above three conditions and examined by sucrose. gradient centrifugation. In this experiment, the RNA obtained directly from the crude nuclei are expected to be larger in size than the RNA prepared from the nuclear extracts, if large structure containing rapidly labeled RNA which really exists in the nuclei of this organism could be partially degraded during the course of the purification of nuclei or the extraction of labeled materials from nuclei. As the experimental result shows in Fig. 2, the sedimentation analysis revealed that there was no significant difference between the radioactivity patterns of the RNA samples used, although the RNA from the nuclear extract obtained by the freezing-thwing procedure became somewhat small in size. These results apparently indicate that rapidly labeled nuclear RNA was hardly degraded during the course of the purification of nuclei or the preparation of RNA. (c) Effect of polyvinylsulfate on rapidly labeled structure In the above experiments polyvinylsulfate (PVS) was used as an RNase inhibitor. Figure 3 shows that the size of the rapidly labeled structure extracted from purified nuclei decreased when PVS was omitted from the extraction buffer. The 20S peak in the sedimentation of the nuclear extracts shifted to about 10S when the extract was prepared in the absence of PVS. This result was also supported by the evidence that the sedimentation coefficient of the RNA of the nuclear extract prepared with the buffered solution without PVS was smaller than that of the RNA of the nuclear extract obtained in the presence of PVS (10 //g/ml) (Fig. 4). However, an increase of the concentration of PVS (more than 20 //g/ml) resulted in the dissociation of the larger structure (25—503) to the 15—20S structure. The P VS effect could also be confirmed by the observation that a high concentration of PVS (20 ^g/ml) stimulates the dissociation of the ribosome to the smaller subunit particles (Fig, 5). These data show that PVS is available as an RNase inhibitor for the extraction of the structure containing rapidly labeled nuclear RNA but destroys the structure when used at a high concentration (more than 20 //g/ml). (d) Effect of Mg2+ on the size of nuclear RNP The effect of magnesium ion concentration on the size of the nuclear RNP particles con-. taining rapidly labeled RNA was examined. As shown in Fig. 6, the yield of the particles decreased with an increase of magnesium ion concentration of the extraction solution, and the. (117).

(17) Fusayuki KANDA. ^10 ^. IQ 7^07. ^o~. bottom Fraction Na toP. "Q.. bottom. bdttom Fractton Na top. 10 .20.. 30. Fraction No.. Fig. 2 Sedimentation analysis of the RNA obtained from crude nuclei and of that from the pH 7.0, pH 8.0 and freezing-thawing nuclear extracts. The cells were labeled with [3H]uracil for 30 min in the presence of 120 /^g/ml actinomycin D. a ; RNA from crude nuclei. Nuclei were prepared from the cell homogenate by centrifugation at 1,000 Xg for 10 min for several times. They were suspended in 25 mM Tris-HCl (pH 7.6) contain-. ing 5 mM EDTA and 2.6% SDS and RNA was extracted with a solution of phenol-chloroform-isoamylalcohol. The RNA sample was mixed with unlabeled 26S and 17S rRNA's of D. discoideum as a marker and analysed on 4.8 ml of a 15—30% (w/v) linear sucrose gradient in the solution consisting of 10 mM Tris-HCl (pH7.6),0.1 M. NaCl and 0.05% SDS by centrifugation for 2 hr at 48,000 rev./min and 22°C in a Spinco SW 50 rotor. After centrifugation, the tube. (118).

(18) Nuclear Ribonucleoprotein Particles in D. discoideum. content was fractionated and measured the absorbance at 260 nm. and radioactivity. b, c and d ; RNA from the pH 7.0 (b), 8.0 (c) and freezing-thawing (d) nuclear extract. The extraction of RNA from pH 7.0, pH 8.0 and freezing-thawing nuclear extract was performed by the SDS-phenol procedure (Iwabuchi et al., 1971). The RNA sample was mixed with unlabeled 26S and 17S rRNA's of D. discoideum and centrifuged in 10—25% (w/v) linear sucrose gradients. in 20 mM Tris-HCl (pH 7.6) containing 0.1 M NaCl, 2 mM EDTA and 20 /ng/m\ PVS at 26,000 rev./min and 8°C for 15 hr using a Hitachi RPS 27 rotor. ® —® , absorbance at 260 nm ; 0— —0 , 3H-radioactivity of RNA precipitated with trichloroacetic acid.. .^)1020~. bottom Fraction No.. top. ^Q.. bottom. 10 20 30. Fraction No. toP. Fig. 3 Sedimentation analysis of the nuclear extracts prepared with TNM. buffer at pH 8.0, after labeling of cells with [3H]uracil for 30 min in. the presence of the 120 fi g/ml actinomycin D. The pH 8.0 nuclear extract was obtained from purified nuclei. During the course of the purification of nuclei and the extraction of the nuclear extract, 10 /Ltg/m\ PVS was used as a RNase inhibitor (a). In the experiment of (b), nuclei were purified in the absence of P VS and the nuclear. extract was prepared with TNM (pH 8.0) buffer from which PVS was omitted. The nuclear extract was centrifuged using a Hitachi. RPS 27 rotor at 26,000 rev./min for 14 hr at 4°C in a 10-25% (w/v). linear sucrose gradient containing 2 M sucrose cushion in the bottom. which was made with TNM (pH 8.0) buffer (b) and TNM (pH 8.0) buffer containing 10 //g/ml P VS (a).. (119).

(19) Fusayuki KANDA. "'Q. 10 20 30 bottom Fraction No. toP. f. 1Q ..20.. 30 bottom Fraction No. ' toP. Fig. 4 Sedimentation analysis of the RNA obtained from pH 8.0 nuclear extract prepared in the presence (a) or absence (b) of 10 p. g/ml PVS. The cells were labeled with [3H]uracil for 30 min in the presence of 120 ^ug/ml actinomycin D. The extraction of RNA from the nuclear extract was performed by the SDS-phenol procedure. The RNA prepared from the nuclear extract was mixed with unlabeled 26S and. 17S rRNA's of D. discoideum and centrifuged in 10—25% (w/v). linear sucrose gradients in 20 mM Tris-HCl (pH 7.6) containing 0.1 M. NaCl, 2 mM EDTA and 20 /^g/ml PVS at 26,000 rev./min and 8°C for 15 hr using a Hitachi RPS 27 rotor. ® —0 , absorbance at 260 run ; 0— —0 , "H-radioactivity of RNA precipitated with trichloroacetic acid.. amount of larger particles was also reduced. This result suggests that an appearance of smallsized nuclear particles was not caused by lowering the concentration of magnesium ions.. (e) CsCl density-gradient analysis of nuclear extracts In order to ascertain whether or not the rapidly labeled hnRNA is associated with any proteins, the nuclear extract was analysed in CsCl density-gradient following glutalaldehyde stabilization according to the method of Baltimore and Huang (1968). Figure 7 shows that the particles containing rapidly labeled hnRNA exhibit one major but slightly dispersed peaks besides several heterodisperse minor ones. The major peak showed a density of 1.62 g/cm3, which is higher than that of mammalian RNP (,pmax=lAO to 1.43 g/cm3) (Georgiev & Samarina, 1971 ; Pederson, 1974a). Therefore, the protein content of this RNP particle was estimated to be 38.3% on the basis of Spirin's formula (Protein %= (1.85-^)/0.006). Thus, it is now very sure that the rapidly labeled nuclear RNA complexes with some proteins to form ribonucleoprotein particles.. (120).

(20) Nuclear Ribonucleoprotein Particles in D. discoideum. ,0^. 20. '0. bottom. bottom. Fraction No,. 10 20. Fraction No.. 30. top. Fig. 5 Sedimentation analysis of the cytoplas-. Fig. 6 Sedimentation analysis of the pH 8.0. mic soluble fraction prepared in the. nuclear extract prepared with TNM. presence of 20 /j.g/m\ PVS. The cells. buffer containing 1 mM, 5 mM or 10. were labeled with [3H]uracil for 30. mM magnesium acetate, after labeling. of cells with [3H]uracil for 30 min in. min followed by treatment with 120/n g /ml actinomycin D. The cells were. the presence of the 120 /ng/ml actino-. lysed in the buffer containing 10 mM Tris-HCl (pH 7.6), 25 mM KC1, 3 mM. mycin D. The purified nuclei were. suspended in the TNM buffer (pH 8.0). magnesium acetate and 20 //g/ml PVS.. containing 1 mM, 5 mM or 10 mM. The cell lysate was centrifuged at 3,000. magnesium acetate. It was then stirr-. rev./min for 10 min and the supernatant. ed for 10 min and centrifuged at. rev./min for 10 min in a ft 1 rotor of a. supernatant fluid was layered onto the. Kubota centrifuge. After centrifuga-. top of a 10—25% (w/v) linear sucrose. fluid was further centrifuged at 10,000. 10,000 Xg for 10 min. The resultant. tion the supernatant fluid (cytoplasmic. gradient containing 2 M sucrose. soluble fraction) was layered onto the. cushion at the bottom which was made. top of a 15—30% (w/v) linear sucrose. with the buffer containing 10 mM Tris-. gradient containing 2 M sucrose. HC1, 0.1 M NaCl and the three different. cushion at the bottom and then centri-. concentrations of magnesium acetate. fuged at 24,000 rev./min at 4°C for 10.5. and then centrifuged at 26,000 rev./min. and 4°C for 14 hr using a Hitachi RPS. hr in an SW 25.1 rotor of a Spinco L ultracentrifuge.. 27 rotor.. , absorbance at 260 nm.. • —® , 1 mM magnesium acetate ; 0— —0 , 5 mM magnesium acetate ; A— —A , 10 mM magnesium acetate.. (121).

(21) Fusayuki KANDA. '0. bottom. 10 20 Fraction No.. 30. top. Fig. 7 CsCl density-gradient analysis of nuclear RNP particles extracted with pH 8.0 TNM buffer. The nuclear RNP containing [3H]uracil-labeled RNA was obtained according to the procedure as described for Fig. 1. The 3H-labeled materials were fixed in 6% neutralized glutalaldehyde and centrifuged in a preformed CsCl densitygradient at 37,000 rev./min (SW 50 rotor) for 17 hr at 4°6°C.. % —% , 3H-radioactivity.. (f) Characterization ofRNA from nuclear RNP particles As already shown in Fig. 2, the sucrose gradient analysis of the RNA prepared from pH 7.0, pH 8.0 and freezing-thawing nuclear extracts directly indicated that almost all of the labeled RNA is heterodispersedly distributed in the region from 5S to 303. The labeled RNA from the pH 7.0 or 8.0 nuclear extract exhibited a count peak in the 15S region, whereas the RNA from the freezing-thawing extract was distributed in a relatively narrow region as a peak at 10S. This indicates that the freezing-thawing method results in the partial degradation of the rapidly labeled nuclear RNA.. It is known that in this organism the rate of synthesis of ribosomal RNA (rRNA) decreases remarkably in the early stage of morphogenesis and the actinomycin D concentration (120 p. g/ml) used here selectively inhibits rRNA synthesis (Firtel et al., 1973 ; Mizukami & Iwabuchi, 1970b ; Egyhazi, 1974). The 28S and 19S ribosomal precursor RNA were scarcely synthesized under the experimental conditions used in this study (Fig. 2).. (122).

(22) Nuclear Ribonucleoprotein Particles in D. discoideum. 0.3. s. 0.2 -? 0.1. s. bottom. 10 20.. 30, Fraction No.. '0.. boitom. 10 20 30.. Fraction No. toP. 10 20 ^. Fraction No. toP. Fig. 8 Sedimentation analysis of RNA of various sizes of rapidly labeled RNP particles prepared from the pH 8.0 nuclear extract. The pH 8.0 nuclear extract was centrifuged using a Hitachi RPS 27 rotor at 26,000 rev./min for 14 hr at 4°C in a 10—25% (w/v) linear sucrose gradient containing 2 M sucrose cushion in the bottom, as described in the regend to Fig. 1. RNA was extracted from the fractions indicated with Roman numerals in Fig. Ib by the SDS-phenol procedure (a, from fraction I ; b, from fraction II ; c, from fraction HI). The RNA was mixed with unlabeled 26S and 17S rRNA's of D. discoideum and then centrifuged in 10-25% (w/v) linear sucrose gradient of 20mM Tris-HCKpH 7.6) containing 0.1M NaCl, 2mM EDTA and 20^g/ml PVS at 26,000 rev./min and 4°C for 15 hr using a Hitachi RPS rotor. H —• , absorbance at 260 nm ; 0— —0 , 3H-radioactivity of RNA precipitated with trichloroacetic acid.. (e) Relationship of the size between nuclear RNA and RNP It is predictable that the rapidly labeled RNA contained in nuclear RNP consists of various sizes of hnRNA's. If one assumes that the high sedimentation velocity of the nuclear structure containing rapidly labeled RNA is due to the amount of proteins distributed evenly along the RNA chain, the broad distribution of the RNP structure which has already been shown in Fig. 1 would be evident for the existence of various sizes of the RNA chain. Thus, the RNA of rapidly sedimenting RNP particles will be surely larger than that of slowly sedimenting ones. To see the relation of the size of RNP and RNA, RNA was extracted from various sizes of the RNP particles and examined in sucrose gradients. Figure 8 shows the sedimentation pattern of rapidly labeled RNA in the RNP particles with various sedimentation coefficients from the pH 8.0 nuclear extract. It has seen in Fig. 8 that 30-50S, 19-29S and 10-17S RNP particles contain 15-30S, 9—23S and 4—15S RNA, respectively. Similar results were obtained in the case of RNP from the pH 7.0 or freezing-thawing nuclear extract (Figs. 9 and 10). Therefore, it is apparent that size of the RNP particles in the nuclear extracts is proportional to that of the RNP contained in the particles ; the mean size of the RNA from the rapidly sedimenting RNP particles was larger than that of the RNA from the slowly sedimenting ones. It must be noted here that the rapidly sedimenting RNP particles occasionally contain 26S. (123).

(23) Fusayuki ,KANDA. ^07 10 20 30. rf0. 10 ,20.. 30. bottom Fractbn No. top. bottom Fraction hto toP Fig. 9. Sedimentation analysis of RNA of two different sizes of RNP partides prepared from the pH 7.0 nuclear extract. The pH 7.0 nuclear extract was centrifuged using a Hitachi RPS 27 rotor at 26,000 rev./ min for 14 hr at 4°C in a 10—25% (w/v) sucrose gradient containing 2 M sucrose cushion in the bottom. RNA was extracted from the fractions indicated with Roman numerals in Fig. la by the SDSphenol procedure (a, from fraction I ; b, from fraction II). The RNA was mixed with unlabeled 26S and 17S rRNA's of D. discoideum and then centrfuged in 10—25% (w/v) linear sucrose gradient of 20 mM Tris-HCl (pH 7.6) containing 0.1M NaCl, 2mM. EDTA and 20//g/ml PVS at 26,000 rev./min and 4°C for 15 hr using a Hitachi RPS 27 rotor. •—® , absorbance at 260 nm ; 0 - -0 , 3H-radioactivity of RNA precipitated with trichloroacetic acid.. "Q. 10 .20. 30. bottom Fraction hk>. t°P. bottom. 1Q ..20,. 30,. rractk)n No. top. Fig. 10 Sedimentation analysis of RNA of two different sizes of RNP partides prepared from the freezing-thawing nuclear extract. The freezing-thawing nuclear extract was centrifuged in a 10—25% (w/ v) sucrose gradient for 14 hr at 26,000 rev./min and 4°C using a Hitachi RPS 27 rotor. RNA was extracted from the fractions indicated with Roman numerals in Fig. 1c by the SDS-phenol procedure (a, from fraction I ; b, from fration II). The RNA was mixed with unlabeled 26S and 17S rRNA's of D. discoideum and then centrifuged in 10—25% (w/v) linear sucrose gradient of 20 mM. Tris-HCl (pH 7.6) containing 0.1M NaCl, 2mM EDTA and 20/^/ml PVS at 26,000 rev./ min and 4°C for 15 hr using a Hitachi RPS rotor. ®—® , absorbance at 260 nm ; 0- -0 , 3H-radioactivity of RNA precipitated with trichloroacetic acid.. (124).

(24) Nuclear Ribonucleoprotein Particles in D. discoideum. 0 bottom. 10 20 30. Fraction No. top. Fig. 11 Comparison in the radioactivity profile between the RNA's obtained from the total nuclear extract and the pH 8.0 extracts. The RNA of various sizes of RNP particles prepared from the pH 8.0 nuclear extract was centrifuged in sucrose gradient as described in Fig. 8. After centrifugation, the gradient was fractionated, absorbance at 260 nm and radioactivity of each fraction were measured. The sum of the 3H-radioactivity of each of the fractions in Fig. 8 I —8 III was. then plotted (solid line). 0 - - 0 , 3H-radioactivity of RNA obtained from the total nuclear extract with pH 8.0 ; ®—® , the sum of 3H-radioactivity of RNA obtained from RNP with various sedimentation coefficients of the pH 8.0 nuclear extract.. ribosomal RNA and 28S ribosomal precursor RNA (cf. Figs. Ib and 81). However, the quantity of the ribosomal RNA's was less than 10% of the total amount of labeled RNA. It is also shown that the sedimentation pattern of the RNA from the pH 8.0 nuclear extract fits well with the pattern of the total count of each fraction in Figs. 81—8m (Fig. 11, solid line). This result suggests that the RNA fron the nuclear extract which has already been shown in Fig. 2 almost represented the RNA of 10 to 50 S RNP particles and further that the RNA did not degrade during the course of sedimentation analysis of RNP particles and the extraction of RNA. (h) Sedimentation analysis of poly(A)-contaming RNA An attempt was made to learn whether or not the RNA contained in the nuclear RNP particle. (125).

(25) Fusayuki KANDA. 70. 90^. time (min). Fig. 12 Effect of the irradiation time on the retention of labeled RNA on poly(U)-filter. The cells were labeled with C3H) uracil for 30 min in the presence of 120 /^g/ml actinomycin D. The RNA of the cytoplasmic soluble fraction was extracted with a phenol-chloroform-isoamylalcohol mixture. The radioactive. RNTA was dissolved in 2 ml of binding buffer (0.1 M sodium phosphate, 0.12 M NaCl, 0.1 mM EDTA, 10 mM Tris-HCl and. 0.5% SDS, pH 7.5) and bound to poly(U)-fiberglass filters which had been irradiated for the indicated time intervals.. The filters were washed according to the following two different methods. In one method, the filters were washed with. 20 ml of binding buffer, followed by 20 ml of ice-cold 5% trichloroacetic acid and 10 ml of 95% ethanol (TCA-ethanol. method, Sheldon et al., 1972). In the other method the poly(U)-filter associated with poly(A)-containing RNA was. washed with 20 ml of binding buffer followed by 20 ml of. ice-cold 0.3 M ammonium acetate and 50% ethanol (Ammonium acetate-ethanol method, Firb et at., 1972). 0 — —0 , TCA-ethanol method ; •—% , Ammonium acetate-ethanol method.. (126).

(26) Nuclear Ribonucleoprotein Particles in D. discoideum. Table IV. Extent of the retention of radioactive RNA containing poly(A) sequence on glass fiber filters under various conditions. TCA-ethanol. method. %. %. 100.0. 210.9. 100.0. 11.2. 4.8. 23.6. 11.2. 67.6. 29.3. 149.8. 80.0. 3.1. 1.3. 2.7. 1.3. 230.7 no irradiation. irradiation -poly(U). method. [3H]count. [3H] count Total. Ammonium-ethanol. The cytoplasmic poly(A)-containing RNA was extracted with phenolchloroform-isoamylalcohol mixture from the cytoplasmic soluble fraction of cells labeled with 3H-uracil for 30 min in the presence of the 120 /^g/ml actinomycin D.. The RNA was dissolved in binding buffer (10 mM Tris-HCl, 0.12 M NaCl,. 0.1 mM EDTA and 0.5% SDS, pH 7.6), then filtered at a flow rate of 2 ml/min. through glass fiber filter (Whatman GF/C) or poly(U)-fiber glass filter (Poly(U) immobilized to Whatman GF/C glass fiber filter). Some of the. poly(U)-containing fiber glass filters were been irradiated for 5 min with a germicidal lamp. After the application of the RNA sample, the filters were. washed with 20 ml of binding buffer, followed by 20 ml of ice-cold 5% trichloroacetic acid and 10 ml of 95% ethanol (TCA-ethanol method, Sheldon et al., 1972). Another method was that the filter associated with the RNA sample was washed with 20 ml of binding buffer followed by 20 ml of ice-cold. 0.3 M ammonium acetate and 50% ethanol (Ammonium acetate-ethanol method, Firtel etal., 1972).. is hnRNA which is a precursor of cytoplasmic mRNA. Since it was reported in D. discoideum as well as in other eukaryotes that hnRNA has a poly(A) sequence at the 3'-end (Sheldon et al., 1972; Firtel etal., 1972 ; Firtel & Lodish, 1973), the poly(A)-containmg nuclear RNA was searched by the poly(U)-filter method. Before the analysis of pulsely labeled nuclear RNA, the poly(U) fiberglass-filter method was checked on the following points. Firstly, an optimum irradiation time with a germicidal lamp was determined, when poly(U) was immobilized on fiberglass filters. Figure 12 shows that the irradiation time of 5—10 min is the most effective for the retention of the radioactive RNA on the filters. In the second, Sheldon et at. (1972) used 5% trichloroacetic acid-95% ethanol (TCAethanol method) to wash the poly(U)-filter to which labeled cytoplasmic RNA had bound, whereas Firtel et al. (1972) (Firtel & Lodish, 1973) used 0.3 M ammonium acetate-50% ethanol (ammonium acetate-ethanol method). As a result of the comparison of the two methods, the ammonium acetate-ethanol method was superior to the TCA-ethanol method. In the third, whether the high retention of the radioactive RNA on the poly(U)-filter is not due to the non-specific binding of RNA to the filter and/or to the molecular aggregation of the radioactive RNA and poly(U), such matter was ascertained by the examination of the amount of the RNA trapped on the glass fiber filter not bearing poly(U). As the data are summarized in Table IV, only 1.3% of the radioactive. (127).

(27) Fusayuki KANDA. 8. b. a. 175. 1~1. ^6 "^ x. E. ^ £'. 3. CM. '&. 265. T. x. &u. ^. <s^. 9. K. E2 Q.'. CF fr. <aL. 1. 6. 2. \\!- 3 _10 20. 6H bdltom Fraction'No. toP. 26S. ^. 10. 175. y-. v. y. Sc,. 0. '] \ \. X. \\ • 61. ^ : sM 4 \} '^ •. ,PO. <aL. lv \ QO°. "5_ _ ..10.. botfom Fraction" No. top. Fig. 13 Sedimentation analysis of poly(A)-containing RNA obtained from total nuclei (a) and cytoplasmic soluble fraction (b). Cells were. labeled with C3H) uracil for 30 min in the presence of 120 jng/m\ actinomycin D. The cytoplasmic soluble fraction and nuclei were obtained as described in "Materials and Methods". Poly(A)-contain-. ing RNA was prepared by the method of Firtel et al. (1972) (Firtel & Lodish, 1973). The cytoplasmic soluble fraction and nuclei were. suspended in 25 mM Tris-HCl (pH 7.6) containing 5 mM EDTA and 2.6% SDS and RNA was extracted with a solution of phenol-chloroform-isoamylalcohol. The RNA sample was mixed with unlabeled 26S and 17S rRNA's of D. discoideum as a marker and analysed on linear sucrose gradients in the solution consisting of 10 mM Tris-HCl. (pH 7.6), 0.1 M NaCl and 0.05% SDS by centrifugation. After centrifugation, the tube content was fractionated and a half portion of each fraction was used for the measurement of the absorbance at 260 nm and radioactivity. The remaining a half portion was ad-. justed to 10 nM of Tris-HCl (pH 7.6), 0.12 M NaCl, 0.1 mM of EDTA and 0.5% SDS at the final concentration and then passed through. poly(U)-filters (poly(U) immobilized to Whatman glassfiber filter, GF/C) prepared according to the procedure described by Sheldon et al. (1972). a, RNA from total nuclei ; b, RNA from cytoplasmic soluble fraction. % ——— ® , H-radioactivity of RNA precipitated with trichloroacetic acid ; 0 — —0 , 3H-radioactivity of RNA bound to poly(U)-filters.. (128).

(28) Nuclear Ribonucleoprotein Particles in D. discoideum. Fig. 14 Comparison of nuclear and cytoplasmic poly(A)-containing RNA's. Cells were labeled for 30 min with C3H) uracil in the presence of 120 //g/ml actinomycin D. The cytoplasmic soluble fractions and nuclei were obtained as described in "Materials and Methods". The nuclear or cytoplasmic poly(A)-containing RNA was extracted as described in the legend to Fig. 13 and in "Materials and Methods". •—® , rapidly labeled nuclear RNA bound to poly(U)filters ; 0——0, cytoplasmic RNA bound to poly(U)filters.. cytoplasmic RNA applied to the filter was retained on the filter without poly(U), whereas about 80% of the RNA was trapped on poly(U)-immobilized filters. Figure 13 shows the sedimentation pattern of all RNA and poly(A)-containing RNA obtained from the cytoplasmic and nuclear fractions (Figs. 13a and 13b). As shown in Fig. 13b, the sedimentation pattern of poly(U)-bindmg radioactive nuclear RNA after pulse-labeling closely resembled that of the total nuclear RNA. Similar results were also obtained with the rapidly labeled RNA from the pH 7.0, pH 8.0 and freezing-thawing nuclear extracts. The distribution pattern of nuclear poly(A)-containing RNA was almost similar to that of cytoplasmic poly(A)containing RNA, although the former was slightly larger in size than the latter (Fig. 14). This result is in accordance with the report by Firtel et al. (1972) who observed that the nuclear precursors of mRNA were somewhat larger than the cytoplasmic mRNA containing poly(A) sequences.. Thus, the above data suggest that almost all the RNA contained in the nuclear RNP particles of the interphase amoebae is certainly hnRNA.. (129).

(29) Fusayuki KANDA. Molecular Weight (xK®. 2 ----4 --g—Q' ,0. Q2 43'^. Migration (cm). Fig. 15 Densitometric tracings of stained gels after electrophoresis of proteins from the nuclear RNP particles of three different preparations on SDS-polyacrylamide gels. The pH 7.0, pH 8.0 and freezing-thawing nuclear extracts were centrifuged in a sucrose gradient as described in "Materials and Methods". After centrifugation, the fractions corresponding to 15—20S were pooled and 0.1 volume of cold 100% (w/v) trichloroacetic acid added. The precipitated materials were collected by centrifugation at 12,000 Xg for 20 min at 0°C. The pellet was dried and then dissolved in 0.1 ml of 0.1 M sodium phosphate (pH 7.0) containing 1% SDS, 1% 2-mercaptoethanol and 4 M urea. It was incubated for 3 hr at 37°C and electrophoresed. on 10% polyacrylamide gels containing 1% SDS for 12 hr at a constant current of 5 mA/gel. For the determination of the. molecular weights of proteins, several marker proteins were parallely run in separate gels. After electrophoresis, gels were stained with coomassie blue, destained with 7% acetic acid and densitometrically scanned at 570 nm as described. (130).

(30) Nuclear Ribonucleoprotein Particles in D. discoideum. previously (Kanda et al., 1974 ; Kanda, 1978). a, proteins of RNP extracted at pH 7.0 ; b, proteins of RNP extracted at pH 8.0 ; c, proteins of RNP extracted by the freezing-thawing method.. Molecular weight (xl<54) 4 6 8__10. in. -^- —^ ^ —,Q Migration (cm). Fig. 16 Densitometric tracings of stained gels after electrophoresis of proteins from the ribosomal fraction on SDS-polyacrylamide gels. The pH 7.0 and pH 8.0 nuclear extracts were centrifuged in a sucrose gradient as described in "Materials and Methods". After centrifugation, the fractions corresponding to 40S ribosomat subunits were pooled and 0.1 volume of cold 100% (w/v) trichloroacetic acid added. The precipitated materials were collected, dried and dissolved in 0.1 ml of 0.1 M sodium phos-. phate (pH 7.0) containing 1% SDS, 1% 2-mercaptoethanol and 4 M urea. It was then incubated and electrophoresed as detailed in the regend to Fig. 15. a, proteins of 40S region in the pH 7.0 nuclear extract ; b, proteins of 40S region in the pH 8.0 nuclear extract.. (131).

(31) Fusayuki KANDA. Molecular wei^it (x \(5A). 4 68 10. 9876543210. Migration (cm). Fig. 17 Densitometric tracings of stained gels after electrophoresis of proteins from the nuclear soluble fraction on SDS-polyacrylamide gels. The pH 7.0, pH 8.0 and freezing-thawing nuclear extracts were centrifuged in a sucrose gradient. After centrifugation, the two fractions from the top of the gradient were pooled and 0.1 volume of. cold 100% (w/v) trichloroacetic acid added. The precipitated. materials were collected, dried and dissolved on 0.1 ml of 0.1 M sodium phosphate buffer (pH 7.0). It was then incubated and electrophoresed as detailed in the legend to Fig. 15. a, proteins of the nuclear soluble fraction of the pH 7.0 nuclear extract; b, proteins of the nuclear soluble fraction of the pH 8.0 nuclear extract ; c, proteins of the nuclear soluble fraction of the freezingthawing nuclear extract.. (i) Protein components of nuclear RNP particles in different preparations The protein moiety of RNP particles was examined by SDS-polyacrylamide gel electrophoresis (Kanda, 1977a). Fig. 15 shows a densitometric tracing of stained gels after electrophoresis of proteins which were prepared from the RNP particles sedimenting in the 15-20S region. Figures 15a, 15b and 15c show the protein patterns of the RNP particles obtained from the pH 7.0, pH 8.0 and freezing-thawing methods, respectively. Two major protein components were commonly present in the 15—20S RNP particles, in addition to several minor ones, irrespective of the difference of procedure for the preparation of nuclear extracts. The molecular. weights of the two major proteins were 42,000 and 60,000 daltones, when estimated from their migration distance relative to some protein markers. The molecular weights of the common. minor proteins were 80,000, 66,000, 47,000, 32,000 and 18,000 daltones. Since the sedimentation coefficient of the small ribosomal subunit of D. discoideum is approximately 40S, the 40S ribosomal subunit will be probably a little contaminated into the 15—20S. (132).

(32) Nuclear Ribonucleoprotein Particles in D. discoideum. Molecular Weight (>. 8 10. Migration (cm) Fig. 18 Densitometric tracing of the stained gel after electrophoresis of proteins from the cytoplasmic soluble fraction on the SDS-polyacrylamide gel. The cytoplasmic soluble fraction was obtained as described in "Materials and Methods". To the cytoplasmic soluble. fraction 0.1 volume of cold 100% (w/v) trichloroacetic acid was added. The precipitated materials were then collected by centrifugation at 12,000 Xg for 20 min at 0°C. The pellet was dried and then dissolved in 0.1 ml of 0.1 M sodium phosphate (pH 7.0) containing 1% SDS, 1% 2-mercaptoethanol and 4 M urea. It was incubated for 3 hr at 37°C and electrophoresed on 10% polyacrylamide gels containing 1% SDS. After electrophoresis, gels were stained with coomassie blue, destained with 7% acetic acid and densitometrically scanned at 570 nm.. RNP fraction. As shown in Fig. 16, the electrophoretic pattern of the proteins obtained from RNP particles sedimenting in the 40S region after sucrose gradient centrifugation of pH 7.0 (Fig. 16b) and pH 8.0 (Fig. 16a) nuclear extract appeared to be similar to that of 408 ribosomal proteins (Kanda etal., 1974). However, since three major components of 40S ribosomal proteins with the molecular weight of 22,000, 16,000 and 38,000 were not found in the protein sample from the 1520S RNP particles, it seems certain that the protein sample from the 15-20S RNP particles was hardly contaminated by the 40S ribosomal proteins. It seemed likely that the 15-20S RNP particles are contaminated by both the nuclear or cytoplasmic soluble proteins. As shown in Fig. 17, the soluble nuclear proteins remaining near the top of the sucrose gradient exhibited a different band pattern from the protein pattern of the RNP particles. Figure 18 shows the densitometric pattern of the total cytoplasmic proteins which consisted of about 15 major protein bands. The proteins with the molecular weight of 10,000-40,000 may originate in ribosomal proteins, because of the agreement of the distribution pattern of the size between two protein species. Other major proteins had molecular weights of 47,000 and 70,000.. (133).

Table I. Effect of Mg2+ concentration on the stability of nuclei.
Tabl II. Yields in various steps during the preparation of nuclei Fraction ODgOo x 103 % Total cell 207 100.0 Crude nuclei 189 91.3 Purified nuclei 143 69.1
Fig. 1 Sedimentation analysis of the nuclear extracts prepared with TNIVt buffer at pH 7.0 (a) and pH 8.0 (b) and by the freezing-thawing method (c), after labeling of cells with [3H]uracil for 30 min in the presence of the 120 jug/mi actinomycin D
Fig. 2 Sedimentation analysis of the RNA obtained from crude nuclei and of that from the pH 7.0, pH 8.0 and freezing-thawing nuclear extracts.
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