Enrichment of selected active human gene sequences in the placental deoxyribonucleic acid fraction associated with tightly bound nonhistone chromosomal proteins

A DNA fraction which is highly enriched in active gene sequences and tightly associated with a subset of nonhistone chromosomal proteins has been isolated from human placenta. After extraction with 2 M NaCl, placental chromatin was separated into two distinct components by centrifugation. Of the total DNA, approximately 96% (DNA-S) is protein free, while the remaining 4% (DNA-P) is tightly complexed with nonhistone chromosomal proteins. Reassociation studies revealed that the DNA-P fraction was enriched 22-fold in actively transcribed human placental lactogen gene sequences, while the DNA-S fraction was correspondingly depleted 22-fold in these sequences. Approximately 45% of the sequences present in DNA-P (equivalent to 1.8% of the genome) were not present in the DNA-S fraction. Reassociation of nick-translated DNA-P to DNA from a partial digest of DNase I treated nuclei indicated that 27% of the DNA-P sequences were DNAase I sensitive, suggesting they may represent actively transcribed gene sequences. Analysis of the overall sequence organization of DNA-P showed that relative to unfractionated DNA and DNA-S, DNA-P was enriched in single-copy sequences, slightly enriched in the class of middle repetitive sequences from C0t 0.01 to 100 M.s, devoid of the more highly repetitive sequences (C0t less than or equal to 0.01). The distribution of total active placental genes between DNA-P and DNA-S was measured by hybridization with a complementary DNA probe transcribed from total polysomal poly(A+) messenger RNA. We found that 57% of this cDNA probe reassociated to DNA-P and 58% to DNA-S, while 95% reassociated to DNA-P mixed with DNA-S at the observed ratio of 4 to 96, suggesting that the DNA-P fraction contained a different population of active gene sequences than DNA-S. From these results we estimate that approximately 85% of the transcribed sequences appear to be distinctly distributed and equally proportioned between DNA-P and DNA-S, while approximately 15% of the transcribed sequences are common to both fractions. We suggest that the strong affinity of the tightly bound nonhistone chromosomal proteins for the DNA-P fraction indicates a likely role for these proteins in the regulation of gene expression.     

Solubility of palmitoyl-coenzyme A in acyltransferase assay buffers containing magnesium ions

The solubility of palmitoyl-CoA is strongly affected by Mg2+ concentrations commonly used in acyltransferase reactions. In 0.10 M Tris-HCl buffer at pH 7.4 or 8.5, all of the palmitoyl-CoA in 10 microM solutions and 90% of the palmitoyl-CoA in 100 microM solutions are precipitated by 1 mM Mg2+. In 0.05 M phosphate at pH 7.4, and in 0.10 M Tris-HCl containing 0.4 M KCl, the substrate remains soluble at Mg2+ concentrations below 4-5 mM. Above 5 mM Mg2+, palmitoyl-CoA is insoluble in all of these buffers. Substrate solubility could therefore be a limiting factor when free Mg2+ and fatty acyl-CoAs are present together during acyltransferase assays.     

M BAND PROTEIN : Two Components Isolated from Chicken Breast Muscle

M band protein can be specifically extracted from fresh chicken breast muscle myofibrils suspended in 5 mM Tris-HCl pH 8.0. During discontinuous polyacrylamide gel electrophoresis the isolated protein separates into three bands which can be identified as two separate components (A, B) and a complex of the two. When partially purified fractions of the separated components are combined, an increase in the intensity of the band containing the complex can be shown. The polypeptide chain weights of the two components are 100,000 (A) and 40,000 (B) daltons as estimated by sodium dodecyl sulfate- (SDS-) polyacrylamide gel electrophoresis. Antibody prepared against total M band protein stains only the M band of the myofibril and is completely absorbed by M band protein. M band protein also absorbs the M band staining specifically from antibody which stains both I and M bands. Immunodiffusion data indicate that anti-M band is a mixture of two specific antibodies, one against each component.     

Characteristics of DNA isolated from a complex form of DNA-polymerase alpha from the rat liver

A complex from of DNA polymerase alpha was isolated from the nuclear membrane of hepatocytes. DNA fragments were shown to be among components of the complex under study. In this paper we present evidence that DNA from the alpha-polymerase complex from quiescent hepatocytes (DNA-G) differs in its nucleotide composition from its counterpart (DNA-S) isolated from hepatocytes synthesizing DNA. As judged by dot hybridization, DNA-G0 does not contain nucleotide sequences which are complementary to ribosomal or messenger RNA, whereas the abovementioned sequences are present in DNA-S. At the same time DNA-G0 is found to contain sequences which are homologous to both SV40 DNA and yeast TRPI-ARS1 DNA. The difference in nucleotide sequences between DNA-G0 and DNA-S indicates that in the process of replication DNA is being stretched across the multienzyme complex located on the nuclear membrane.     

Fractionation of desmosomes and comparison of the polypeptide composition of desmosomes prepared from two bovine epithelial tissues

Desmosomes isolated from bovine tongue mucosa or muzzle epidermis appeared identical by ultrastructural analyses but had some differences in their polypeptide compositions as determined by SDS-PAGE. These preparations were extracted in 9 M urea, 10 mM Tris-HCl (pH 9), and 25 mM B-mercaptoethanol and then centrifuged at 240,000g for 30 min. The urea-soluble and insoluble fractions were analyzed by SDS-PAGE. The urea soluble fractions of both tongue and muzzle desmosomes were enriched in polypeptides of 240, 210, 81, and 75 kDa and also polypeptides (40 to 70 kDa) that were keratin-like, as determined by immunoblotting analyses with keratin antisera. The urea insoluble fraction of tongue desmosomes contained glycoproteins of 165, 160, 140, 110, and 100 kDa, while this fraction from muzzle contained glycoproteins of 165, 115, and 105 kDa. Ultrastructural examinations of insoluble pellets obtained from urea extracted tongue and muzzle desmosomes showed that most of the components at the cytoplasmic faces of the desmosomes were removed, while the membrane regions of the desmosomes resisted the treatment. The urea soluble proteins were dialyzed against 10 mM Tris-HCl (pH 7.6), and the resulting preparation was pelleted by centrifugation and examined by electron microscopy. Ultrastructural examination of this material revealed that it had assembled into a fibrillar meshwork, similar to the fibrillar region adjacent to the submembranous plaque of isolated desmosomes. Thus, treatment of isolated desmosomes with 9 M urea allowed the fractionation of membrane-associated desmosomal proteins from cytoplasmic desmosomal proteins. A comparison of these fractions from tongue and muzzle indicated that the polypeptide compositions of the desmosomes varied between tissues, especially with respect to the fractions enriched in either glycoproteins or keratin.     

Rapid isolation of eukaryotic DNA

Guanidine hydrochloride (GHCl) is frequently used for the isolation of RNA from eukaryotic cells. However, it appears that its use for the rapid isolation of chromosomal DNA has been little appreciated. Intact and readily digestible DNA was prepared from a range of murine tissues and cell lines which had been dispersed in GHCl and ethanol precipitated, and then rinsed in 70% ethanol and resuspended in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5. This simple technique is ideally suited to the preparation of DNA from large numbers of tissue samples.     

Tightly-bound form of poly(ADP-ribose)polymerase in the higher order of chromatin organization

A tightly-bound form of poly(ADP-ribose)polymerase is present, within the third level of rat testis chromatin structure, both in the loops and in chromatin matrix. When chromatin matrix was extensively digested with DNAaseI, only little residual enzymatic activity remained in the insoluble fraction, the extent of DNA hydrolysis being well correlated to the progressive loss of the poly(ADP-ribose)polymerase activity. These findings suggest that the tightly-bound form of the enzyme is not an intrinsic protein component of chromatin matrix but is only indirectly located in this structure, being rather associated to the attachment points of loop DNA on the matrix.     

Partial purification of human parathyroid hormone 1-84 as a thioredoxin fusion form in recombinant Escherichia coli by thermoosmotic shock

A modified purification method, thermoosmotic shock (osmotic shock coupled with heat-treatment) for heat-stable proteins, was devised in the purification of Trx-hPTH (1-84) (human parathyroid hormone coupled with thioredoxin as a fusion partner) from E. coli. Thermoosmotic shock can integrate the functions of extraction and crude separation of fusion protein Trx-hPTH (1-84). To improve the purification efficiency, thermoosmotic shock conditions were optimized and achieved as follows: the optimized high osmotic solution containing 20mM Tris-HCl buffer (pH 8.0), 1mM EDTA, and 25% sucrose; the low osmotic solution containing 20mM Tris-HCl buffer (pH 8.0), 1mM EDTA, and the heat-treatment temperature of 100 degrees C for 10 min. Using this method, the purity of Trx-hPTH (1-84) was up to 73% and the yield was up to 72%, respectively. In addition, the two separation methods of both thermoosmotic shock and affinity chromatography have been compared, indicating that thermoosmotic shock is an economical and feasible way for the fusion protein separation. Besides, the thermoosmotic shock method may be used for the purification of some proteins of thermal stability without N-terminal His-tag.     

A new DNA binding mode for CAP

In the absence of cyclic AMP, the Escherichia coli cyclic AMP receptor protein (CAP) binds without detectable sequence specificity to restriction fragments containing lac and crp promoter sequences. Under standard conditions (10 mM Tris, 1 mM EDTA, pH 8.0), our estimates of the equilibrium constant and cooperativity parameter for complex formation are 114,000 +/- 1400 M-1 and 1.3 +/- 0.8, respectively. Thus, this interaction lacks the substantial cooperativity previously reported for CAP binding to genomic DNAs. Using the electrophoresis mobility shift assay, we find that complexes of increasing CAP content differ by a highly uniform mobility decrement. This result is most consistent with a binding mode in which little or no DNA bending occurs. The ability of CAP to distinguish between restriction fragments and genomic DNA, shown by the difference in binding cooperativity, suggests the existence of previously unsuspected DNA sequences or structures that modulate its binding cooperativity.     

A rapid alkaline extraction procedure for screening recombinant plasmid DNA.

A procedure for extracting plasmid DNA from bacterial cells is described. The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.     

Effect of salts and chromatin concentrations on the buoyant density of chromatin in metrizamide gradient.

Using isopycnic centrifugation in metrizamide gradient, effect of ions and chromatin concentration on the buoyant density of chromatin was quantitatively examined. An elevation followed by gradual decline and secondary increase of the density occurred in accordance with increase in MgCl2 or NaCl concentration. Maximum density was observed at a concentration of these salts known to result in the condensation of chromatin. Release of protein occurred during the phase of density decline. The second increase in density is mainly due to the density increment of DNA in the chromatin. The density was dependent upon the concentration of chromatin in a band formed in the metrizamide gradient, while the density of free DNA and protein was not so greatly affected by their concentration. The density of chromatin in the presence of 0.14 M NaCl was less affected by the chromatin concentration than that in the absence of salt. Calculation of results indicates that grade of hydration of chromatin at concentrations lower than 400 microgram/ml in 1 mM Tris-HCl (pH 8.0) is higher than that expected from its DNA and protein components.     

Mg2+-dependent unwinding of DNA by nonhistone chromosomal protein HMG(1 + 2) from pig thymus as determined by DNA melting temperature analysis

In the previous studies with endonucleases specific for single-stranded DNA, we have indicated that the nonhistone chromosomal protein HMG(1 + 2) prepared from pig thymus has an activity to unwind DNA partially at low protein-to-DNA weight ratios (Yoshida, M. & Shimura, K. (1984) J. Biochem. 95, 117-124). In the present work, we have pursued the unwinding reaction by HMG(1 + 2) by thermal melting temperature analysis of DNA, and by investigating the effect of Mg2+ on the reaction. The melting temperature of DNA in the presence of HMG(1 + 2) at low protein weight ratios decreased in 2 mM Tris-HCl, pH 7.8, whereas it increased at higher ratios. The depressions of melting temperature by HMG(1 + 2) at low ratios were not observed either in the system of 2 mM Tris-HCl, pH 7.8, containing EDTA or in the system containing samples treated in advance with EDTA. An addition of Mg2+ to the system reproduced the depression of melting temperature at low protein-to-DNA ratios as well as the increase at higher ratios. Analysis by Mg2+-equilibrated gel filtration revealed that HMG(1 + 2) is a Mg2+-binding protein. However, the depression of melting temperature at low protein-to-DNA ratios was not due to removal of Mg2+ from DNA by HMG(1 + 2). From these results, it is concluded that HMG(1 + 2) causes a partial DNA unwinding detectable by thermal melting temperature analysis of DNA, and that Mg2+ is necessary for the unwinding reaction.     

Mitochondrial adenosine triphosphatase from human placenta--purification and catalytic properties

1. The purification of ATPase (EC 3.6.1.3) from human placental mitochondria is described. The yield based on mitochondrial enzyme activity was about 70% and the purification was 380-fold. 2. The rate of Mg-ATP hydrolysis was 85 mumole per min per mg of protein under optimum conditions. 3. Nucleoside triphosphates were hydrolyzed by the purified enzyme at decreasing rates in the following order: GTP greater than ITP greater than ATP greater than epsilon-ATP greater than UTP greater than CTP in Tris-HCl buffer (pH 8.0), and in the order: ATP greater than GTP greater than or equal to ITP greater than epsilon-ATP greater than UTP greater than CTP in Tris-bicarbonate buffer at pH 8.0. 4. The values of kinetic parameters are reported. The ATPase reaction deviated from typical Michaelis-Menten kinetics in Tris-HCl buffer but not in Tris-bicarbonate. Eadie-Hofstee plots for Mg-ATP hydrolysis were biphasic in Tris-HCl (Km = 0.2 mM, 0.09 mM) and monophastic in Tris-becarbonate medium (Km = 0.16 mM). 5. In the presence of Mg-ITP or Mg-GTP as substrates no curvature of the reciprocal plots was observed. 6. The results presented reflect the fact that multiple conformations of the enzyme molecule do exist and are probably involved in its regulatory functions. 7. The existence of two kinetically distinct classes of catalytic sites and of an anion-binding site on the placental ATPase is proposed.     

Solubilization of chromatin by an endogenous enzymic Ca2+, Mg2+-dependent factor. Activity of residual chromatin]

An endogenous Ca2+, Mg2+-dependent factor of enzymic nature (apparently an endonuclease) digests a part of chromatin in the rat liver nuclei producing DNA fragments of an uniform size. After 60 min of incubation at 15 degrees C and pH 7.50 in the presence of 5 mM MgCl2 and 2 mM CaCl2 87-93% of the total chromatin becomes soluble. The insoluble chromatin however contains 70-85% of the in vivo newly synthesized RNA. In regenerating liver the proportion of the insoluble residual chromatin increases while the radioactivity of the newly synthesized DNA in this fraction is highest. Residual chromatin can be solubilized by ultrasonic treatment only. The Ca2+, Mg2+-dependent dissolving factor is not present either in brain or in PMN leucocyte nuclei.     

The non-histone proteins of the rat liver nucleus and their distribution amongst chromatin fractions as produced by nuclease digestion.

The search for proteins involved in maintaining higher order chromatin structures has led to a systematic examination of the non-histone proteins (NHP) of rat liver nuclei in the context of nuclease digestion studies. 40-45% of the 3H-tryptophan labelled NHP originally present could be removed by extensive washing in a "physiological" buffer, incubation at 37 degrees C with or without nuclease and a further wash step. Nuclei at this stage had a remarkably constant NHP content (ca. 0.73 micrograms/micrograms DNA), independent of the degree of digestion with micrococcal nuclease or HaeIII. The solubilized chromatin produced by limited digestion with either nuclease contained 0.3-0.5 microgram NHP/microgram DNA, this value falling to ca. 0.16 after more extensive cleavage. Insoluble chromatin fractions were between 2-fold (very limited digestion) and 16-fold (extensive digestion) richer in NHP than the corresponding soluble fractions. Gel electrophoresis revealed about 12 NHP bands in soluble fractions, the most prominent of M.Wt. 41.400, while the insoluble material had at least 50 components. These properties were independent of whether lysis of nuclei occurred in 0.2 or 50 mM ionic strength. The large disparity in NHP content between complementary soluble and insoluble chromatin fractions is considered in terms of chromatin organization in vivo and the possible role of NHP migration.     

Ammonia affects the glycosylation patterns of recombinant mouse placental lactogen-I by chinese hamster ovary cells in a pH-dependent manner

The N-linked glycosylation of the recombinant protein mouse placental lactogen-I (mPL-I) expressed by Chinese hamster ovary (CHO) cells under nongrowth conditions was inhibited by increasing levels of ammonium chloride (3 and 9 mM) in a serum-free, protein expression medium. The effect of ammonia on glycosylation was dependent on the extracellular pH (pH(e)). In media containing 0 and 9 mM ammonium chloride, the percentage of the most heavily glycosylated forms of secreted mPL-I decreased from ca. 90% to ca. 25% at pH(e) 8.0, and from ca. 90% to ca. 65% at pH(e) 7.6, respectively. However, at pH(e) 7.2, the most heavily glycosylated forms of secreted mPL-I decreased from ca. 90% to ca. 80% in media containing 0 and 9 mM ammonium chloride, respectively. Inhibition of mPL-I glycosylation was found to correlate with the calculated concentrations of the ammonia species (NH(3)). Control experiments showed that the ammonia effect on mPL-I glycosylation could not be attributed to increased chloride concentration or osmolarity, or to extracellular events after secretion of the recombinant protein into the supernatant. Ammonium chloride, 9 mM, inhibited the expression rate of MPL-I by CHO cells at low pH(e). (c) 1994 John Wiley & Sons, Inc.     

The use of Haemophilus gallinarum DNA-relaxing enzyme to investigate the relationship between the number of superhelical turns and the molecular weight in a negatively twisted DNA.

Covalently closed-circular, superhelical DNAs, including viral DNAs, bacterial plasmid DNAs, and bacteriophage replicative-form DNA, were treated with a small amount of Haemophilus gallinarum DNA-relaxing enzyme to generate incompletely relaxed DNA molecules. Each sample consisted of a set of closed-circular DNA molecules differing by one turn in their number of superhelical turns. The DNA samples were analyzed by agarose gel electrophoresis under conditions such that the electrophoretic mobility was a function of the number of turns. The numbers of superhelical turns (at 37 degrees C in 20 mM Tris-HCl (pH 7.5)-5 mM MgCl2) in the DNAs of pSC101 (5.8 megadaltons), Colicin E1 (4.2 megadaltons), pMR4 (4.0 megadaltons; recombinant between pBR322 and lambda DNA fragment), phi X174 replicative-form (RF) I, Simian virus 40 (SV40), and polyoma virus (3.4--3.6 megadaltons each), and lambda dv021 (2.05 megadaltons) were estimated to be 36, 27, 23--24, 20--21, 20--21, 20--21, and 11--13, respectively. It appears that the number of superhelical turns is mainly a function of the molecular weight of the DNA, at least in the substrates tested here.     

A protein that accumulates during starvation in Tetrahymena nuclei

Tetrahymena pyriformis was starved in 50 mM Tris-HCl, pH 7.5, at 28 degrees C. The number of cells did not change appreciably under the starvation conditions. Nuclear proteins of unstarved cells and cells starved for 1, 2, 4, and 7 d were analyzed by SDS-polyacrylamide gel electrophoresis. Most of the large amount of nonhistone proteins present in the unstarved cell nucleus disappeared with the starvation time. However, the relative amounts of the high mobility group protein and histones did not change appreciably. On the other hand, a protein with a molecular weight of ca. 16,000 gradually accumulated in the nucleus on starvation. This protein was extracted with 0.25 M HCl, but was not soluble in 0.5 M perchloric acid. The amino acid composition and molecular weight of this protein were similar to those of HMG protein LG-2 of T. thermophila. Some lysyl endopeptidase peptides of this protein were found to have amino acid sequences present in LG-2, thus we tentatively named it an LG-2-like protein.     

Polymerization of actin induced by a molar excess of ATP in a low salt buffer.

The polymerization of actin induced by dilution has previously been reported, where a 1000-fold molar excess of ATP over actin resulted when actin was diluted to 4.0 micrograms/ml in low salt buffer A (0.1 mM ATP, 0.1 mM CaCl2, 2 mM Tris-HCl, pH 8.0, 5 mM 2-mercaptoethanol, 1 mM NaN3). Filaments formed by the addition of ATP to a 1000-fold molar excess over actin in buffer B (0.1 mM CaCl2, 2 mM Tris-HCl, pH 8.0, 1 mM NaN3) were then separated by gel-filtration. When ATP was removed from these filaments using Dowex-1, depolymerization occurred. Thus, the reversible polymerization induced by the dilution of actin or by addition of ATP can be ascribed to the binding of ATP at the low affinity site of actin.