NUCLEAR PROTEINS OF THE GUINEA PIG BRAIN

Abstract—

• 1 Chromatin protein fractions were separated from the nuclei from brain, liver and kidney of the guinea pig. The fractions were studied by electrophoretic methods and amino acid analysis.
• 2 Brain nuclear fractions were washed with 0.15m -NaCl and nuclear acidic proteins then removed by 0.35 m -NaCl. These 0.35 m -NaCl-extracted proteins were considered to be similar to the nuclear soluble acidic proteins.
• 3 Nonhistone-1, histone and nonhistone-2 fractions were obtained from 2.0 m -NaCl-soluble chromatin fractions by lowering the salt concentration and successive extraction with acid and alkali. The nonhistone-3 fraction was also extracted from the nuclear residue by alkaline solution.
• 4 The contents and characteristics of the nonhistone fractions of the brain, especially the nonhistone-1 fraction, differed among the three tissues. The histone fractions showed no obvious difference among the three tissues. The nonhistone-1 fraction of the brain, which comprised a low percentage of total nuclear protein, contained relatively high amounts of acidic proteins.

     

The disc electrophoretic separation of proteins from various parts of the guinea pig brain

—Disc electrophoresis was used to study the saline-soluble and detergent-soluble proteins of various parts of the auditory pathway in the guinea pig brain. The five areas studied were the cochlear nucleus, olivary complex, inferior colliculus, medial geniculate and the auditory cortex. A study was also made of the proteins extracted from subcellular fractions obtained from guinea pig cerebral cortex. The electrophoretic patterns showed small but significant differences in the five areas of the brain, both in the fast-running acidic proteins and in the very slow-moving proteins. The higher levels of the auditory pathway, to which the more complex information processing and storage is normally attributed, showed more complex electrophoretic patterns than the lower areas. Gross differences also occurred in the patterns of both the saline-soluble and T-X-100-soluble proteins of the subcellular fractions obtained by gradient density centrifugation. The simplest patterns were obtained from the myelin-rich fraction and the mitochondrial fraction whilst the most complex patterns were given by the proteins of the nerve ending fraction and the cell soluble fraction.     

NONHISTONE NUCLEAR PROTEINS OF RAT BRAIN

Abstract— The rat brain was dissected into cerebral cortex, cerebellum and the remaining regions. From the nuclei, isolated from these three brain sections, were extracted two fractions of nuclear sap proteins (proteins soluble in 014 M NaCl and proteins soluble in 01 M Tris-HCl buffer pH 7-6) and two fractions of nonhistone chromosomal proteins (one soluble in 0-35 M NaCl and one which is not soluble at this salt concentration). Each of these four fractions of the nonhistone nuclear proteins was further separated by polyacrylamide gel electrophoresis. The electrophoretic patterns of the studied fractions of nuclear proteins are qualitatively identical regardless of the brain section from which the analysed protein fraction was isolated. In addition, there arc no qualitative differences in the electrophoretic patterns of nonhistone chromosomal proteins which are and which are not soluble in 0-35 M NaCl. In contrast to the qualitative similarity of the electrophoretic patterns of proteins from different sections of the brain, the amount of the nonhistone nuclear proteins is characteristic for each studied brain section. The ratio of the total nonhistone nuclear proteins to DNA is highest in the brain cortex and lowest in the cerebellum. The most expressed difference between the nuclei is in the ratio of the nonhistone chromosomal proteins soluble in 0-35 M NaCl to DNA. This ratio is 0-52 in the cortex. 0-38 in the mixture of noncortical and noncerebel-lar regions and only 0-18 in the cerebellum. The amount of the three fractions of nonhistone nuclear proteins in the nuclei of individual brain sections is proportional to the activity of the genome in these nuclei. The only exception are the nonhistone chromosomal proteins which are not soluble in 0-35 M NaCl. These proteins and the histones are present in the same amounts in nuclei isolated from all three studied sections of the brain. The results support a proposal that the nonhistone nuclear proteins are involved in the expression of the genetic activity of the cell, without the majority of the proteins in any of the four fractions being the specific regulatory molecules.     

Proteinase activity of nuclei from various tissues towards endogenous histones]

The proteinase activities of nuclei isolated from tissues differing in their mitotic activities (brain, thymus, liver, ascite lymphoma) towards the histones and non-histone acid -- extractable proteins were studied. The sensitivity of different histone fractions to nuclear proteinase depends on temperature and time of nuclei incubation under conditions providing for complete dissociation of chromatin proteins from DNA (2 M NaCl--5 M urea). The proteinase activity in the brain and thymus nuclei is revealed only under prolonged (43 hrs) incubation of the nuclei at 25 degrees C, which is accompanied by partial proteolysis of histone H1. Histone H4 from brain nuclei and histone H2a from thymus nuclei are preferably degraded. In the nuclei isolated from the mice ascite cell lymphoma NK/ly and from rat liver the enzyme activity is revealed mainly towards the arginine-enriched histones H3 and H4. The proteolysis of the arginine-enriched histones in tumour cell nuclei is more complete. A high sensitivity to proteolysis was observed for non-histone acid-extractable proteins with low electrophoretic mobility, which were found in brain and tumour cell nuclei.     

Nuclear fraction enriched in interchromatin granules

A fraction enriched in interchromatin granules (IG) has been obtained from isolated liver nuclei by digestion with DNase I and extraction with salt solutions. The fraction contains interchromatin granules in the form of conglomerations and some contaminating material of nucleolar and nuclear matrix origin. The ultrastructure of the fraction enriched in IG and electrophoretic patterns of proteins are described.     

ANALYSE ELECTROPHORETIQUE COMPAREE DES PROTEINES CEREBRALES TOTALES, SOLUBLES ET PARTICULAIRES, CHEZ CINQ SOUCHES DE SOURIS

Abstract—

• 1 The distribution of total cerebral proteins from five strains of adult mice: Quaking (Qk), C57BL/6, CBA, DBA/2 and C57BR have been compared according to various subcellular brain fractions. The saline or detergent-soluble proteins were separated in a pH discontinuous system by analytical disc electrophoresis on polyacrylamide gel, either at pH 8·9 (acidic proteins) or pH 4·3 (basic proteins).
• 2 Significant quantitative differences and higher electrophoretic mobility were observed both in the high molecular weight acidic proteins of myelin and in the low molecular weight soluble proteins of myelinic and synaptosomal fractions of the Quaking mutant. Differences between Qk and control were inapparent in nuclear, mitochondrial or microsomal protein fractions.
• 3 The in vivo incorporation of tritiated amino acids into the brain proteins of the Qk mouse have been studied. An increased level of incorporation was found in two soluble, acidic proteins of the supernatant cell sap.
• 4 The electrophoretic patterns of the brain proteins from three other inbred strains (CBA, DBA/2 and C57BR) were identical except DBA/2, whose soluble and acidic proteins of the supernatant cell sap were characterized by two supplementary minor bands. The observed protein abnormalities have been discussed in relation to the alteration of the CNS myelination and to the genetic lesions presumed to be responsible for a cellular multienzymic induction.

     

Analysis of age-associated alteration in the synthesis of HMG nonhistone proteins of the rat liver

HMG proteins were extracted with 5% PCA or 0.35 M NaCl from whole tissue, nuclei or chromatin of the liver of young (19 weeks) and old (118 weeks) male rats. They were resolved on acetic acid-urea polyacrylamide gel. The electrophoretic patterns of the major HMG proteins 1, 2, 14 and 17 of both ages are similar. The in vitro synthesis of HMG 1 and 2 decreases, but that of HMG 14 and 17 increases considerably in the liver of old rats. The synthesis of different HMG proteins is modulated differentially by spermine, butyrate, dexamethasone and 3-aminobenzamide in the liver of young and old rats. These findings suggest that HMG proteins contribute to alterations in the organization of chromatin and expression of genes during aging.     

Changes in labeling of soluble and solubilized rat brain proteins using (3H)-leucine as precursor during a learning experiment.

This paper deals with an electrophoretic study on soluble and solubilized rat brain proteins during a brightness discrimination. After intraventricular injection of L-[3H]-leucine the most pronounced increase in relative specific activity was found for several soluble acidic protein-band complexes as well as for solubilized slow-migrating non-myelin proteins obtained from hippocampus of trained animals over the data obtained for active and passive controls.     

Analysis of putative high-mobility-group (HMG) proteins in neuronal and glial nuclei from rabbit brain

Putative high-mobility-group (HMG) proteins 1, 2, and 17 were detected in neuronal and glial nuclei isolated from the cerebral hemisphere of rabbit brain. Although divergent chromatin structures are present in these two populations of brain nuclei (i.e., neuronal nuclei exhibit a short DNA repeat length), no differences were apparent in the electrophoretic mobilities of putative HMG proteins 1, 2, and 17 on SDS gels.     

ELECTROPHORETIC CHARACTERIZATION OF BASIC PROTEINS IN ACID EXTRACTS OF CENTRAL NERVOUS SYSTEM TISSUE

A technique has been outlined for identification of myelin basic proteins in mixtures of CNS proteins. Myelin basic proteins can be recognized easily by high cathodic mobility at low pH, a unique electrophoretic pattern exhibited at high pH and a characteristic colour when complexed with Amido black. The major protein extracted at pH 3·0 from either brain or spinal cord is myelin basic protein. In the low pH electrophoretic pattern of these extracts it is the most conspicuous component and the component migrating farthest cathodically; it does not appear in comparable electrophoretic patterns of liver extracts. Guinea pig myelin basic protein appears as a single dense blue-green band in low pH electrophoretic patterns, in contrast to the other proteins which are stained greyish-blue or greyish-purple by Amido black. The pattern of rat myelin basic protein is similar except that it consists of a pair of dense blue-green bands. A third characteristic which facilitates the identification of myelin basic proteins in mixtures is a considerable cathodic mobility and electrophoretic heterogeneity at pH 10·6. Most other basic CNS proteins barely penetrate the gel at this pH. We have also examined in detail the behaviour of two other components of pH 3·0 extracts which migrate close to myelin basic protein at low pH. Both are present in pH 3·0 extracts of liver and brain but not of spinal cord, and both stain grey instead of blue-green, a characteristic which readily distinguishes them from myelin basic protein. Neither of these components affects the characteristic pattern of microheterogeneity observed in high pH electrophoretograms of myelin basic proteins. One of these components has been purified and tentatively identified as lysine-rich histone F1.     

Polyamine induced changes in the ADP-ribosylation of nuclear proteins from rat liver

Purified rat liver nuclei were incubated $\text{in vitro}$ with [³H]NAD. Altered patterns of ADP-ribosylation of nuclear proteins occurred with 1 mM spermidine or spermine with the latter polyamine causing the greater change. Spermine treated nuclei showed a two-fold increase in ADP-ribose incorporation into H1 histones and a decrease in the other histones. Likewise, the incorporation into the more acidic non-histone nuclear proteins was greater with spermine than spermidine. These results suggest that polyamines may exert a regulatory function by altering the pattern of ADP-ribosylation of both histone and non-histone nuclear proteins.     

High mobility group (HMG) proteins in the tammar wallaby Macropus eugenii: quantitative variations between tissues and testis-specific co-extracted proteins

1. Tammar wallaby (Macropus eugenii, Marsupialia) proteins with similar electrophoretic mobilities to calf non-histone chromosomal proteins HMG 1, 2, 14 and 17 are perchloric acid extracted from whole tissues (liver, kidney, spleen, brain and testis) and purified liver nuclei (using PCA or 0.35 M NaCl). 2. Tammar and calf HMG 1 have similar amino acid compositions. 3. Two testis-specific basic proteins co-extracting with HMG-like proteins from both tammar and red kangaroo (Megaleia rufa) are found in whole testis, purified testis nuclei, but not epididymis. 4. Tammar HMG 2 separates into two components on both acid urea and SDS gels. The larger, more basic protein, HMG 2b, is relatively abundant in proliferating tissues (testis, spleen).     

大鼠衰老过程中脑细胞DNA与c－Ha－ras原癌基因的甲基化

用ＭｓｐⅠ／ＨｐａⅡ酶解电泳法和高效液相色谱（ＨＰＬＣ）两种方法进行比较,研究了不同年龄大鼠的肝、脑细胞基因组ＤＮＡ的甲基化程度。从酶解电泳图谱可观察到,肝、脑细胞基因组ＤＮＡ甲基化在青年鼠和老年鼠之间没有差异。但用具有高分辨率的高效液相色谱测量ＤＮＡ中５－ｍＣ的含量时发现,老年鼠脑细胞ＤＮＡ甲基化程度较大年鼠的下降６２％,而肝细胞ＤＮＡ甲基化程度在老年鼠与青年鼠之间并没有显著差异。这些结果提示：（１）用常规的酶解电泳法所分析的ＤＮＡ甲基化结果并不能反映整个基因组ＤＮＡ甲基化的水平。（２）衰老过程中,不同组织ＤＮＡ甲基化的改变存在差异,引起这种差异的原因可能与组织的增殖和分化程度有关。进一步分析脑细胞原癌基因ｃ－Ｈａ－ｒａｓ的甲基化水平,无论ＭｓｐⅠ酶切图谱,还是ＨｐａⅡ酶切图谱均可观察到分子大小为１９ｋｂ、７．５ｋｂ、１．３ｋｂ、０．９ｋｂ的四条阳性带,说明该基因未发生甲基化,且与年龄无关。     

An altered ribosomal protein in an edeine-resistant mutant of Saccharomyces cerevisiae

Summary The r-proteins of an edeine-resistant mutant of Saccharomyces cerevisiae were compared to those of the wild-type strain by using two different two-dimensional electrophoretic techniques: (1) the Kaltschmidt-Wittmann method and, (2) the Kaltschmidt-Wittmann system, in the first dimension and the Na Dodecyl-SO₄ system in the second.With the first technique, the results indicate that the patterns of basic ribosomal proteins are similar in the two strains. However, the pattern of acidic ribosomal proteins of the mutant revealed an additional protein band with respect to the normal one. Using the other technique, the patterns of basic and acidic ribosomal proteins of the mutant demonstrated a similarity to the corresponding pattern of the wild-type strain.The data disclose that an acidic ribosomal protein of the mutant may have two forms with different electrophoretic mobilities and similar molecular weights.     

Comparative study of lymphocyte non-histone proteins

The non-histone chromosomal proteins of bovine lymphocytes were investigated by the two dimensional gel electrophoresis of O'Farrell. The 0.35 M NaCl extractable proteins from lymphocyte nuclei, the high mobility group proteins (HMG) and some proteins released from nuclei by DNase I were compared on the basis of their electrophoretic patterns.     

Isolation of nuclear acidic proteins from rat tissues. Characterization of acetylated liver nuclear acidic proteins

Nuclear acidic proteins isolated from rat brain, heart, kidney and liver showed similar, complex patterns on electrophoresis in sodium dodecyl sulphate-polyacrylamide gels. The contamination of nuclear acidic proteins by nuclear-membrane acidic proteins was found to the extent of 11%. Incorporation of [(3)H]acetate into the various nuclear acidic proteins in vivo, which were fractionated by polyacrylamide-gel electrophoresis, differed from tissue to tissue. Hydrolysis of these acetylated nuclear acidic proteins with 6m-HCl at 110 degrees C released 70% of the radioactivity, which indicated that labile acetyl groups had been incorporated into these proteins. Analysis of [(3)H]acetate-labelled nuclear acidic proteins revealed two acetylated amino acid residues, N(2)-acetylserine and N(2)-acetyl-lysine. The significance of the role played by nuclear acidic proteins in relation to gene regulation is discussed.     

ELECTROPHORETIC ANALYSIS OF THE HIGHLY BASIC PROTEINS OF THE RAT BRAIN FRACTION WHICH INDUCES EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS

Abstract— A purified basic protein fraction of adult rat brain when injected into guinea pigs induced experimental allergic encephalomyelitis (EAE). The freeze-dried preparations were subjected to electrophoresis on 5% polyacrylamide gel at pH 10.6 in the presence of 8 m -urea to obtain one-step separation of highly basic proteins from other proteins. Under these conditions the highly basic proteins whose isoelectric point exceeded pH 10.0 gave seven distinct components. After staining these protein bands with naphthalene black 10B they were scanned densitometrically: the area under each peak was computed and used for calculation of the amounts of individual basic proteins. The acid extracts of rat brain contained 2.61–3.95 mg highly basic proteins/g fresh tissue.
A comparison of the electrophoretic patterns of acid extracts of rat brain and liver showed that two of the highly basic proteins (components 1 and 2) were present only in the brain and not in the liver. These two components in the brain were attributed to proteins of the myelin sheath.     

COMPARATIVE ELECTROPHORESIS AND ISOZYME ANALYSIS OF SEED PROTEINS FROM CULTIVATED RACES OF SORGHUM

Comparative disc electrophoresis of acidic proteins, basic proteins, and isozymes of esterase, MDH, and peroxidase were performed with aqueous extracts of seeds from seven cultivars belonging to five races of Sorghum bicolor ssp. bicolor: bicolor, caudatum, durra, guinea, and kafir. Two disc electrophoretic systems were employed. Acidic proteins were electrophoresed in an anionic system (tris-glycine buffer, pH 8.3). Basic proteins were electrophoresed in a cationic system (β-alanin-acetate buffer, pH 4.5). Soluble proteins were stained with Coomassie brilliant blue. Isozyme activity was detected by using specific enzyme stains and substrates. Each cultivar yielded reproducible, characteristic patterns of distinct acidic and basic proteins. Cultivars belonging to the same race produced identical protein and isozyme patterns. The degree of electrophoretic similarity among races was estimated by calculating similarity index values for each of the 10 possible pairs of races. Bicolor, caudatum, durra, and guinea produced very similar acidic and basic protein patterns and esterase, MDH, and peroxidase isozyme patterns. Differences, however, were observed among all races. All of kafir patterns were significantly different from the patterns of other races. Comparative electrophoresis may provide a new source of taxonomic characters for investigating phenetic and phylogenetic relationships in Sorghum.     

Changing patterns of nucleic acids, basic and acidic proteins in generative nuclei during pollen germination and pollen tube growth in Hippeastrum belladona

Generative and vegetative nuclei of mature and germinated pollen grains from Hippeastrum belladonna were separated in a continuous Ficoll gradient. Less than 3% contamination was observed between the generative and vegetative nuclear fractions. The vegetative nuclei were composed of two populations; the larger population consisted of nuclei with 1C levels of DNA and the smaller with 2C levels. The generative nuclei consisted of a homogeneous population composed of nuclei possessing 2C levels of DNA. Histone synthesis did not occur in vegetative nuclei. Changes appeared in the gel-electrophoretic banding patterns of the F1 histones of vegetative nuclei during germination. Changes were not observed in the generative nuclei. A reduction of general proteins and RNA was observed in vegetative nuclei by 20 h of germination. The phenol-soluble nuclear proteins of vegetative nuclei revealed transitions in electrophoretic banding patterns during pollen germination that were greater than those shown by the histones. These changes in the PSNP primarily involved reduced concentrations of certain proteins rather than synthesis of new ones. However, a new band was observed in the electrophoretic pattern of the PSNP of vegetative nuclei after 12 h of pollen tube growth. No transition was seen in the PSNP of generative nuclei during pollen germination and tube growth. The regulatory role of the PSNP in cell differentiation is discussed in the light of these findings.