Quantitative characterization of specific genomic promoters using agarose gel electrophoresis

Over the past decade a large number of studies have focused attention on the role of nucleosomes as negative and positive regulators of specific nuclear functions. Due to the lack of an analytical method to determine the higher order conformation of the nucleosomal arrays that encompass specific genetic loci (e.g., promoters, enhancers), research emphasis has mostly been centered on chromatin remodeling and histone posttranslational modifications. We have recently developed an agarose gel electrophoresis method that permits us to analyze the higher order structure of specific in vivo assembled chromatin fragments. After calibration using a well-defined in vitro system, we have been able to experimentally determine the size, shape, and conformational flexibility of the Mouse Mammary Tumor Virus long-terminal repeat promoter region in its repressed and activated states. These studies pave the way for widespread analyses of the higher order structure of specific, functionally important chromosomal loci, and in so doing enhance our understanding of the roles that the higher order structure of chromatin play in genome regulations.     

Purification and some properties of a deoxyribonucleic acid endonuclease endogenous to rat liver chromatin

A deoxyribonucleic acid (DNA) endonucleolytic activity has been purified from a 0.3 M KCl extract of rat liver chromatin by a combination of selective precipitation and ion-exchange and gel filtration chromatography. The purified protein has a molecular weight of 35 000 as determined by Sephadex G-200 gel filtration and sodium dodecyl sulfate-acrylamide gel electrophoresis. The nuclease activity is stimulated by the addition of Mg2+ and thus may represent the Mg2+-activated DNase endogenous to chromatin. The purified enzyme has the ability to make both single-strand nicks and double-strand cuts in DNA.     

Minichromosome of simian virus 40: presence of histone HI.

In contrast to conclusions of previous studies /I-3/ claiming the absence of histone HI from the SV40 and polyoma viral minichromosomes we have found that a preparation of purified SV40 minichromosomes does contain histone HI. The content of HI in relation to other four histones in the SV40 minichromosomes is close to that in the cellular chromatin. Histone HI in the isolated SV40 minichromosomes is bound apparently to internucleosomal DNA stretches as was shown already for HI in the cellular chromatin /4/. In addition it was found that more than 90% of the purified SV40 minichromosomes migrated as a single discrete deoxyribonucleoprotein band upon agarose gel electrophoresis.     

Purification and Characterization of Clathrin from Bovine Brain

Abstract: Clathrin has been purified to electrophoretic homogeneity by initial extraction of clathrin from purified coated vesicle fraction, followed by column chromatographies with gel filtration. DEAE-cellulose, and hydroxylapatite and finally by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Antibody specific to clathrin has also been obtained. Two forms of native clathrin, fast and slow components, have been prepared to about 95% purity by hydroxylapatite column chromatography. Both fast and slow components are believed to represent two different aggregates of clathrin subunit because they comigrate in agarose electrophoresis. pH 7.4, and also migrate as clathrin subunit on SDS-PAGE with a molecular weight of 175,000. Furthermore, both components cross-react with antibody against purified clathrin and compete for antibody binding site with labeled fast component. The fast component can also be converted to the slow component. In addition to clathrin, two proteins of about 38,000 and 35,000 M.W. that consistently co-purified with native clathrin are probably also intrinsic to coated vesicle.     

Purification of a new high activity form of glucose-6-phosphate dehydrogenase from rat liver and the effect of enzyme inactivation on its immunochemical reactivity.

A new form of cytoplasmic glucose-6-phosphate dehydrogenase (E.C.1.1.1.49) was purified from rat liver by protamine sulfate precipitation, ammonium sulfate fractionation, ion exchange chromatography with diethylaminoethyl cellulose, and affinity chromatography with Cibacron blue agarose and NADP agarose. This form of the enzyme has a specific activity of over 600 units/mg of protein and gives essentially a single band by polyacrylamide gel electrophoresis. The form of the enzyme isolated by this purification method is 3 times more active than the form purified from liver by previously reported procedures. The relative mass of this pure glucose-6-phosphate dehydrogenase enzyme was determined by disc gel electrophoresis to be 269,000. This high activity glucose-6-phosphate dehydrogenase enzyme, after inactivation by reaction with palmityl-CoA, was no longer precipitated by specific rabbit and goat antisera to this purified enzyme. Thus, the possibility still exists that starved fat-refed animals contain glucose-6-phosphate dehydrogenase (G6PD) enzyme protein in an inactivated form no longer detectable by either enzyme activity or immunoprecipitation.     

Semisynthetic β-lactam antibiotics synthesizing enzyme from Acetobacter turbidans: purification and properties

Semisynthetic cephalosporin synthesizing enzyme has been purified from cell-free extract of Acetobacter turbidans ATCC 9325 by ion-exchange, hydrophobic chromatography and gel filtration. The purified enzyme migrated as two bands on SDS-gel electrophoresis and as six bands on native gel electrophoresis. This enzyme has an isoelectric point at 5.8 and contains most of the essential amino acids. The molecular weight was estimated to be 280 000 to 290 000 by gel filtration. Two different subunits of this enzyme having molecular weights of 70 000 and 72 000 have been identified in the presence of sodium dodecyl sulphate. The purified enzyme favours the synthetic reaction over the hydrolytic reaction by a factor of 2.6 times, as determined by the ratio of relative activities.     

Purification and characterization of putative endothelin converting enzyme in bovine adrenal medulla: evidence for a cathepsin D-like enzyme

A specific and sensitive assay has been established for measurement of endothelin converting activity in a tissue extract. This assay is based on measuring endothelin-1 generated from big endothelin-1 by endothelin converting enzyme (ECE) with radioimmunoassay using an endothelin C-terminal specific antibody. By using this assay, we purified and characterized ECE in bovine adrenomedullary chromaffin granules ECE was purified over 3,000 times by a combination of DEAE, hydrophobic and gel filtration chromatography. A molecular weight of ECE was estimated to be approximately 30,000 by gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that ECE had three major components with estimated molecular weights of 45,000, 30,000 and 15,000 like bovine spleen cathepsin D. ECE had a pH optimum at 3.5 and was inhibited by pepstatin. These results strongly suggest that ECE is a cathepsin D-like aspartic protease.     

Cell-free translation of murine coronavirus RNA.

The coding assignments of the intracellular murine hepatitis virus-specific subgenomic RNA species and murine hepatitis virion RNA have been investigated by cell-free translation. The six murine hepatitis virus-specific subgenomic RNAs were partially purified by agarose gel electrophoresis and translated in an mRNA-dependent rabbit reticulocyte lysate, and the cell-free translation products were characterized by gel electrophoresis, immunoprecipitation, and tryptic peptide mapping. These studies have shown that RNA 7 codes for the nucleocapsid protein, RNA 6 codes for the E1 protein, RNA 3 codes for the E2 protein, and RNA 2 codes for a 35,000-dalton nonstructural protein. Genomic RNA directs the cell-free synthesis of three structurally related polypeptides of greater than 200,000 in molecular weight.     

从水稻细胞核中制备分子量达到Mb级的DNA

用水稻黄化苗作实验材料,经蔗糖不连续梯度纯化细胞核、琼脂糖包埋、蛋白酶消化释放DNA,脉冲交变电泳显示所得DNA样品分子量在200kb至3Mb之间,其中大量集中在2.2Mb处.所得DNA容易被多种内切酶消化和连接,可用于构建水稻YAC文库和大尺度物理图的研究.     

Macromolecular complexes of aminoacyl-tRNA synthetases from eukaryotes. 1. Extensive purification and characterization of the high-molecular-weight complex(es) of seven aminoacyl-tRNA synthetases from sheep liver.

Starting from homogenates of sheep liver, extensive co-purification of seven aminoacyl-tRNA synthetases to high specific activities was achieved by a three-step procedure involving fractional precipitation by poly(ethylene glycol) 6000, gel filtration on 6% agarose and chromatography on Sepharose-bound tRNA. The purified material is composed of nine major protein components as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and has an apparent molecular weight of about 10(6) estimated by gel filtration on 6% agarose. It contains aminoacyl-tRNA synthetase activities specific for methionine, lysine, arginine, leucine, isoleucine, glutamine and glutamic acid. The rigorous co-elution of these seven enzymes at each chromatographic step suggests, but does not conclusively prove, that they are physically associated within the same complex. The enzyme composition of the high-molecular-weight complex purified from sheep liver is identical to that of the complex previously isolated from human placenta by Denney in 1977 (Arch. Biochem. Biophys. 183, 156--167).     

Molecular morphology of cyanobacterial phycobilisomes

Phycobilisomes were isolated from several cyanobacteria following cell lysis with Triton X-100. They were purified by phosphate precipitation and hydrophobic-interaction chromatography. Their phycobiliprotein compositions were quantitatively determined by application of sets of simultaneous absorbance equations to gel chromatographic separations of the chromoproteins. Phycobilisomes purified from several cyanobacteria had characteristic elution times on agarose gel chromatography. Combining electron microscope observations of phycobilisome structure, phycobiliprotein composition, and agarose gel chromatography estimates of molecular weight permitted the calculation of many details of phycobilisome molecular structure. Complementary chromatic adaptation resulted in a change of phycobilisome composition and structure. The polypeptide compositions of phycobilisomes were examined by sodium dodecyl sulfate-agarose gel chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The phycobilisomes were composed of phycobilipeptides derived from the constituent phycobiliproteins. Higher molecular-weight phycobilipeptide aggregates were also observed. The dominant forces responsible for the maintenance of phycobilisome structure are concluded to be hydrophobic interactions.     

Asymmetric somatic cell hybridization in plants

As part of an investigation into whether it would be possible to use UV radiation as a suitable pretreatment of the donor cells in asymmetric hybridization experiments, the effects of this treatment on sugarbeet (Beta vulgaris L.) protoplast DNA have been determined and compared with those of gamma radiation. Both nuclear and mitochondrial DNAs have been examined. The dose ranges chosen had previously been determined to be potentially applicable for fusion experiments. Pulsed field gel electrophoresis and standard agarose gel electrophoresis have been used in combination with laser scanning densitometry to gain an insight into the precise nature and degree of DNA damage resulting from irradiation. It was observed that UV radiation introduced substantial modifications to sugarbeet DNA. Double-strand breaks were detected, the number of which was found to be directly proportional to the dose applied. Such breaks indicate that UV radiation results in substantial chromosome/chromatid fragmentation in these cells. Chemical modifications to the DNA structure could be revealed by a significant reduction in DNA hybridization to specific mitochondrial and nuclear DNA probes. Following gamma irradiation at equivalent biological doses (i.e. those just sufficient to prevent colony formation) much less damage was detected. Fewer DNA fragments were produced indicating the presence of fewer double-strand breaks in the DNA structure. In comparison to UV treatments, DNA hybridization to specific probes following gamma radiation was inhibited less. For both treatments, mitochondrial DNA appeared more sensitive to damage than nuclear DNA. The possibility that DNA repair processes might account for these differences has also been investigated. Results indicate either that repair processes are not involved in the effects observed or that DNA repair occurs so fast that it was not possible to demonstrate such involvement with the experimental system used. The general relevance of such processes to asymmetric cell hybridization is discussed.     

Studies on prephenoloxidase-activating enzyme from cuticle of the silkworm Bombyx mori. II. Purification and characterization of the enzyme

Prephenoloxidase-activating enzyme has been purified approximately 4800-fold from cuticular extract of the silkworm, and the preparation seems to be homogeneous as judged by disc- and dodecylsulfate-polyacrylamide gel electrophoresis. By means of gel filtration through Sephadex G-100, it has been supposed that the enzyme exists as mono- and dimeric forms at slightly acidic pH, while a monomeric form is predominant under slightly alkaline condition. The molecular weight of the monomer was estimated to be 33,000–35,000 by dodecylsulfate-polyacrylamide gel electrophoresis and gel filtration.It has been demonstrated that ester substrates for trypsin, benzoyl-l-arginine ethyl ester and tosyl-l-arginine methyl ester, can be hydrolyzed by the purified enzyme. Several lines of evidence indicating that a single protein is involved in both activation and esterolytic reactions have been presented. Some enzymatic properties of the purified preparation as esterase have also been described.In connection to esterase activity of the purified enzyme, a mechanism of prephenoloxidase activation in the silkworm system has briefly been discussed.     

Charting histone modifications and the functional organization of mammalian genomes

A succession of technological advances over the past decade have enabled researchers to chart maps of histone modifications and related chromatin structures with increasing accuracy, comprehensiveness and throughput. The resulting data sets highlight the interplay between chromatin and genome function, dynamic variations in chromatin structure across cellular conditions, and emerging roles for large-scale domains and higher-ordered chromatin organization. Here we review a selection of recent studies that have probed histone modifications and successive layers of chromatin structure in mammalian genomes, the patterns that have been identified and future directions for research.     

A Rapid Purification Procedure for Camel Kidney Glutathione S-Transferase

Glutathione S-transferase was isolated from supernatant of camel kidney homogenate centrifugation at 37, 000 xg by glutathione agarose affinity chromatography. The enzyme preparation has a specific activity of 44 μ;mol/min/mg protein and recovery was more than 85% of the enzyme activity in the crude extract. Glutathione agarose affinity chromatography resulted in a purification factor of about 49 and chromatofocusing resolved the purified enzyme into two major isoenzymes (pI 8.7 and 7.9) and two minor isoenzymes (pI 8.3 and 6.9). The homogeneity of the purified enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration on Sephadex G-100.

The different isoenzymes were composed of a binary combination of two subunits with molecular weight of 29, 000 D and 26, 000 D to give a native molecular weight of 55, 000 D.

The substrate specificities of the major camel kidney glutathione S-transferase isoenzymes were determined towards a range of substrates. l-chloro-2, 4-dinltrobenzene was the preferred substrate for all the isoenzymes. Isoenzyme III (pI 7.9) had higher specific activity for ethacrynic acid and isoenzyme II (pI 8.3) was the only isoenzyme that exhibited peroxidase activity. Ouchterlony double-diffusion analysis with rabbit antiserum prepared against the camel kidney enzyme showed fusion of precipitation lines with the enzymes from camel brain, liver and lung and no cross reactivity was observed with enzymes from kidneys of sheep, cow, rat, rabbit and mouse.

Different storage conditions have been found to affect the enzyme activity and the loss in activity was marked at room temperature and upon repeated freezing and thawing.     

Distribution of tissue-specific tightly bound non-histone proteins in the first level of repeating chromatin structures

Non-histone proteins, tightly bound to DNA, have been extracted from whole chromatin and core particles prepared from pig liver or kidney. We have investigated by bidimensional slab gel electrophoresis the distribution of this protein class in the first level of repeating structure of chromatin. Our results reveal that non-histone proteins tightly bound to DNA are a heterogeneous protein class. Some of them, particularly in the core particles, appear to be essentially the same in both tissues, though having differences in their isoelectric point, which may be attributed to postsynthetic modifications. We have calculated that this protein class is associated to only 10% of nucleosomes, these nucleosomes having, on the average, one protein molecule for each core DNA. The tissue-specific proteins have high molecular mass (ranging from 135 kDa to 70 kDa in liver, over 135 kDa in kidney) and, in kidney, a more basic isoelectric point. These proteins are mainly located outside the core particles; they could be situated in the spacer regions and/or be involved in determining higher levels of chromatin organization.     

A rapid purification procedure for camel kidney glutathione S-transferase

Glutathione S-transferase was isolated from supernatant of camel kidney homogenate centrifugation at 37,000 xg by glutathione agarose affinity chromatography. The enzyme preparation has a specific activity of 44 mumol/min/mg protein and recovery was more than 85% of the enzyme activity in the crude extract. Glutathione agarose affinity chromatography resulted in a purification factor of about 49 and chromatofocusing resolved the purified enzyme into two major isoenzymes (pI 8.7 and 7.9) and two minor isoenzymes (pI 8.3 and 6.9). The homogeneity of the purified enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration on Sephadex G-100. The different isoenzymes were composed of a binary combination of two subunits with molecular weight of 29,000 D and 26,000 D to give a native molecular weight of 55,000 D. The substrate specificities of the major camel kidney glutathione S-transferase isoenzymes were determined towards a range of substrates. 1-chloro-2,4-dinitrobenzene was the preferred substrate for all the isoenzymes. Isoenzyme III (pI 7.9) had higher specific activity for ethacrynic acid and isoenzyme II (pI 8.3) was the only isoenzyme that exhibited peroxidase activity. Ouchterlony double-diffusion analysis with rabbit antiserum prepared against the camel kidney enzyme showed fusion of precipitation lines with the enzymes from camel brain, liver and lung and no cross reactivity was observed with enzymes from kidneys of sheep, cow, rat, rabbit and mouse. Different storage conditions have been found to affect the enzyme activity and the loss in activity was marked at room temperature and upon repeated freezing and thawing.     

Dynamics of rat hepatocyte histone glycoxidation after general x-ray irradiation

Dynamics of structural parameters of hepatocyte histone glucoxidative modification 3, 9 and 24 h after general X-ray irradiation of rats at dose 5 Gy was studied. Dynamics of these parameters (content of carbonyl groups, bityrosyl cross-linkings, pentosidines, advanced glycation end products) was compared with alterations in DNA structure (according to agarose gel electrophoresis) and lipid peroxidation extent (by malondialdehyde content). Oxidative stress induced by hepatocyte irradiation results in structural damage of DNA and histones accompanied by an increase of histone bityrosyl cross-linking and carbonyl content. The content of advanced glycation end products in histones corresponds to the extent to DNA damage and malondialdehyde content. The described postradiation modifications of histones may be important for regulation of chromatin function.