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R L Seale

Publications and source records attributed to R L Seale.

At least 19 recordsLinked to original sources
PubMed1990

Expression, purification, and characterization of human factor XIII in Saccharomyces cerevisiae.

Factor XIII is the terminal enzyme of the clotting cascade. A cDNA sequence encoding human placental factor XIII was expressed in Saccharomyces cerevisiae with the yeast ADH2-4c promoter. Expression levels were a strong function of the noncoding flanking DNA content of the construction. When the terminal 3'-flanking noncoding DNA was removed, expression increased approximately 50-fold. The protein was produced in quantity by high-yield fermentation and purified to homogeneity. The recombinant protein was cleaved by thrombin at the same activation site as purified human placental FXIII and exhibited 100% enzymatic activity. At high thrombin concentrations rFXIIIa was cleaved into inactive 54- and 25-kDa polypeptides. The identity of these cleavage sites and the blocked N-terminus to that of the human protein was revealed by amino acid microsequencing. A time course of thrombin activation was performed and the relative distribution of the thrombin-cleaved subunits to the uncleaved zymogen subunits determined; the results were consistent with the half of the sites catalytic model for transglutaminase activity proposed by Chung et al. (Chung, S. I., Lewis, M. S., & Folk, J. E. (1974) J. Biol. Chem. 249, 940-950, 1974) and Hornyak et al. (Hornyak, T. J., Bishop, P. D., & Shafer, J. A. (1989) Biochemistry 28, 7326-7332). Equilibrium and velocity sedimentation analysis indicated that rFXIII exists as a 166-kDa nondissociating dimer that behaves as a compact particle of 8.02 S. Thus, all of the properties of rFXIII thus far examined are consistent with those reported for human platelet and placental FXIII.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1988

Treatment with sodium butyrate inhibits the complete condensation of interphase chromatin.

The effects of histone hyperacetylation on chromatin fiber structure were studied using direct observations with the electron microscope. Histone hyperacetylation was induced in HeLa cells by treatment with sodium butyrate, and the ultrastructure of control and of acetylated chromatin fibers examined after fixation at different stages of compaction. No differences between control and acetylated chromatin were seen when the fibers were partially unfolded (10 mM NaCl, 20 mM NaCl, 50 mM NaCl), but in 100 mM NaCl, control chromatin showed further compaction to the "30 nm" fiber, while hyperacetylated chromatin failed to undergo this final compaction step. These results strongly suggest that histone acetylation causes a moderate "relaxation" rather than complete decondensation of interphase chromatin fibers. The relationship of these findings to the increased DNase I sensitivity of acetylated chromatin, and to transcription and replication, is discussed.

Acetylation↗
PubMed1984

Presence of nucleosomes within irregularly cleaved fragments of newly replicated chromatin.

In previous reports (Annunziato et al., J. Biol. Chem., 256:11880-11886 [1981]; Annunziato and Seale, Biochemistry 21:5431-5438 [1982]) we have described two classes of newly replicated chromatin which differ in structure, solubility properties, and requirements for maturation. One class is nucleosomal, soluble at low to intermediate ionic strengths, and acquires mature nucleosomal composition and normal repeat length in the absence of concurrent protein synthesis. In contrast, the other class is cleaved irregularly by MNase (appearing as a smear in DNA gels), is insoluble at moderate ionic strengths, requires protein synthesis to gain normal subunit structure, and comprises approximately 60% of total new chromatin DNA after mild nuclease digestion. It is now demonstrated that this heterogeneous component (produced by the action of either MNase or Hae III on chromatin replicated in cycloheximide) yields nucleosomes when redigested with MNase. The presence of nucleosomes within heterogeneous chromatin fragments suggests that nucleosomal and non-nucleosomal regions may be juxtaposed during chromatin replication. These findings are discussed with respect to current models of nucleosome segregation.

Chromatin↗
PubMed1984

beta-Globin gene family in murine erythroleukemia cells resides within two chromatin domains differing in higher order structure.

The beta-globin gene family is organized into two distinct chromatin domains which are digested at significantly different rates by DNase I. We have investigated the possibility that this differential DNase I sensitivity is based upon differences in the higher order structure of chromatin. When nuclei are digested under low ionic strength conditions known to unfold higher order chromatin structures, the differential sensitivity is lost. That is, the relatively DNase I resistant domain, containing the transcriptionally inactive embryonic and beta-homologous globin genes, becomes sensitive. When chromatin is recondensed with either MgCl2 or NaCl, thus indicating the higher order coiling of the chromatin fiber, the differential sensitivity is restored. Furthermore, the removal of histone H1, known to be essential for stabilization of higher order chromatin structures, results in the loss of differential DNase I sensitivity. In contrast to the DNase I resistant domain, the transcriptionally active adult beta-globin genes show no increase in the rate of digestion when chromatin is unfolded, indicating that this domain may exist as an unfolded nucleosomal chain. The data further suggest that this sensitive domain may be depleted of histone H1.

Animals↗
PubMed1984

Chromatin structure of the beta-globin gene family in murine erythroleukemia cells.

We have analyzed the chromatin structure of the beta-major globin gene and other related beta-globin genes in induced and uninduced murine erythroleukemia (MEL) cell nuclei. Nuclei were digested with either DNase I or micrococcal nuclease, and the purified DNA was hybridized to a set of cloned genomic DNA fragments covering the beta-globin gene region. This region consisted of two distinct domains as characterized by sensitivity to DNase I digestion. One domain was relatively sensitive and contained the potentially active or actively transcribed beta-major and beta-minor globin genes. The other, relatively insensitive domain contained the nontranscribed embryonic and beta-globin homologous genes. The sensitivity of these domains was not altered during erythroid differentiation. In nonerythroid cells, the entire globin gene family, including the adult and embryonic globin genes, was contained in a single relatively resistant domain. Micrococcal nuclease (MNase) also defined two general domains of nuclease sensitivity that coincided with those of DNase I. However, the relatively sensitive MNase domain containing the beta-major and beta-minor genes became more sensitive upon chemically stimulated erythroid differentiation. A detailed examination of the beta-major globin gene revealed that the actual coding region became increasingly sensitive to micrococcal nuclease after differentiation while the 5'-flanking DNA did not. Thus, micrococcal nuclease was able to accurately define the primary transcription unit of the beta-major gene.

Animals↗
PubMed1983

Histone deacetylation is required for the maturation of newly replicated chromatin.

The effects of inhibiting histone deacetylation on the maturation of newly replicated chromatin have been examined. HeLa cells were labeled with [3H]thymidine in the presence or absence of sodium butyrate; control experiments demonstrated that butyrate did not significantly inhibit DNA replication for at least 70 min. Like normal nascent chromatin, chromatin labeled for brief periods (0.5-1 min) in the presence of butyrate was more sensitive to digestion with DNase I and micrococcal nuclease than control bulk chromatin. However, chromatin replicated in butyrate did not mature as in normal replication, but instead retained approximately 50% of its heightened sensitivity to DNase I. Incubation of mature chromatin in butyrate for 1 h did not induce DNase I sensitivity: therefore, the presence of sodium butyrate was required during replication to preserve the increased digestibility of nascent chromatin DNA. In contrast, sodium butyrate did not inhibit or retard the maturation of newly replicated chromatin when assayed by micrococcal nuclease digestion, as determined by the following criteria: 1) digestion to acid solubility, 2) rate of conversion to mononucleosomes, 3) repeat length, and 4) presence of non-nucleosomal DNA. Consistent with the properties of chromatin replicated in butyrate, micrococcal nuclease also did not preferentially attack the internucleosomal linkers of chromatin regions acetylated in vivo. The observation of a novel chromatin replication intermediate, which is highly sensitive to DNase I but possesses normal resistance to micrococcal nuclease, suggests that nucleosome assembly and histone deacetylation are not obligatorily coordinated. Thus, while deacetylation is required for chromatin maturation, histone acetylation apparently affects chromatin organization at a level distinct from that of core particle or linker, possibly by altering higher order structure.

Acetylation↗
PubMed1983

High mobility group proteins: abundance, turnover, and relationship to transcriptionally active chromatin.

We have measured the abundance of high mobility group (HMG) proteins 14 and 17 in HeLa cell chromatin and their fractionation with respect to transcriptionally active sequences. HMG protein 17 constitutes 10-20% of the mass of an individual core histone; HMG 14 is approximately one-tenth the mass of HMG 17. The enrichment of HMG proteins, relative to bulk chromatin, is less than 2-fold in the chromatin fraction enriched 6-fold in active sequences. The digestion characteristics of HMG nucleosomes indicate that they are interspersed with H1 nucleosomes and other monomer species. The HMG monomers are quite resistant to degradation by micrococcal nuclease and can be resolved as distinct nucleoprotein entities after trimming of the DNA to core length. Turnover measurements showed that HMG proteins 14 and 17 are stable for at least 24 h. When nucleosome monomers are reconstituted with a 0.35 M NaCl nuclear protein extract, each nucleosome subtype can be reconstituted; however, this is a function of both the amount of extract added and the DNA length of the nucleosomes. When the kinetics of reconstitution of bulk vs. coding sequences were measured with cDNA, there was no significant enrichment of active sequences in the HMG-containing mononucleosomes of HeLa cells at any ratio of extract to monomer employed. In Friend cells, the abundance of sequences among mononucleosome species was the same for the transcribed beta-major globin gene, a transcriptionally inactive embryonic globin, and an inactive immunoglobulin gene. There was little correlation of HMG content with transcriptionally active chromatin, either native or reconstituted.

Chromatin↗
PubMed1983

Chromatin replication, reconstitution and assembly.

Many previously held concepts about the replication of chromatin have recently been revised, or seriously challenged. For instance, within the last two years, evidence has accumulated to indicate that newly synthesized DNA is not the sole site of deposition of newly synthesized histones, and that histones are not only made, but are assembled into chromatin in the absence of DNA synthesis. Furthermore, segregation of parental histones to daughter DNA duplexes may be bidirectional, rather than the previously accepted unidirectional mechanism. The storage of histones prior to assembly apparently involves histone pairs rather than octamers, and similarly, histones associate with DNA in (apparent) pairs, rather than as pre-assembled octameric units. It is currently questioned whether or not nucleoplasmin is involved in either histone storage or nucleosome assembly. The onset of histone synthesis has recently been found to occur in late G1 rather than in S, and thus is independent of DNA synthesis; however, the cessation of histone synthesis is linked to that of DNA. Thus, there emerges from this newly accumulated data the conclusion that chromatin biosynthesis is not as straightforward as was believed just a few years ago. As we review the evidence on each of these subjects, we attempt to point out directions for future experimentation.

Chromatin↗
PubMed1983

Transcribed chromatin exhibits an altered nucleosomal spacing.

The nucleosomal repeat lengths of bulk chromatin and the chromatin of transcriptionally active and inactive genes were analyzed in two mouse cell lines and adult mouse spleens. The adult beta-globin gene exhibits a nucleosomal repeat length approximately 11 base pairs longer than (i) an inactive embryonic globin gene, epsilon y3; (ii) an immunoglobulin heavy chain gene, Cmu; and (iii) the bulk chromatin in murine erythroleukemia cell line DS19. The repeat length of the Cmu gene was approximately 14 base pairs longer than that of the adult beta-globin or epsilon y3 genes in the IgM-producing cell line M104E. The chromatin of several inactive genes had repeat lengths less than or equal to bulk chromatin. Individual genes were shown to vary in repeat length among the cell types examined. In addition, genes that exhibited an increased nucleosomal spacing were digested to mononucleosomes more rapidly than bulk chromatin or inactive genes with shorter repeats. Increased repeat length was also correlated with an increased sensitivity to DNase I. Thus, increased nucleosomal spacing may be a property of transcriptionally active genes or genes with the potential for transcription.

Animals↗
PubMed1982

Maturation of nucleosomal and nonnucleosomal components of nascent chromatin: differential requirements for concurrent protein synthesis.

The DNA of newly replicated chromatin is comprised of two components, distinguishable by their solubility characteristics and requirements for maturation. One of these components possesses core histones, typical nucleosomal structure, a nuclease-resistant core containing 146 base pairs (bp) of new DNA, and all the nucleosomal species found in bulk chromatin (due to bound histone H1 and high mobility group proteins). In addition, this class of nascent chromatin exhibits a shortened repeat length of approximately 165 bp, as opposed to the 288-bp repeat of bulk chromatin. Within 10 min of DNA synthesis, the spacing of mature chromatin is established; the spacing maturation can occur in the absence of protein synthesis. The second class of nascent DNA is distinguished from the nucleosomal component by its insolubility, lack of discernible nucleosomal organization, and dependence on protein synthesis to attain typical subunit structure. This unassembled component is not free DNA, as demonstrated by its intermediate resistance to nucleolytic degradation. The structural properties and maturation requirements of this material suggest that it is the site of de novo nucleosome assembly.

Chromatin↗
PubMed1982

Association of newly synthesized histones with replicating and nonreplicating regions of chromatin.

Histone deposition in HeLa cells has been studied by monitoring the fractionation and electrophoresis mobility of pulse-labeled histones under conditions that separate newly replicated from bulk chromatin DNA. The separation efficiency of these two methods is approximately 70%. Following micrococcal nuclease digestion, chromatin was fractionated by salt elution. 50-65% of the newly synthesized histones eluted with bulk chromatin at NaCl concentrations between 0.1 and 0.3 M and were further down to co-electrophorese with bulk chromatin DNA, not with the more extensively digested newly replicated chromatin DNA contained in those fractions. The remaining chromatin fractions, solubilized with 0.4-0.6 M NaCl, were several-fold enriched in nascent DNA (Annunziato, A. T., Schindler, R. K., Thomas, C. A., Jr., and Seale, R. L. (1981) J. Biol. Chem. 256, 11880-11886) and were correspondingly enriched for the balance (35-50%) of newly synthesized core histones. This fraction of newly synthesized core histone may be preferentially deposited onto newly replicated DNA. In contrast, histone H1 showed little tendency toward deposition onto new DNA. Within 15 min all new core histones attained the same solubility and electrophoretic mobility as bulk chromatin. We conclude that newly synthesized histones are deposited onto both replicating and nonreplicating regions of chromatin.

Chromatin↗
PubMed1981

Dual nature of newly replicated chromatin. Evidence for nucleosomal and non-nucleosomal DNA at the site of native replication forks.

When chromatin is extracted from nuclease-digested nuclei by stepwise salt elution, two different classes of newly replicated chromatin can be distinguished. Nascent DNA eluted from nuclei under conditions of low to moderate ionic strength (0.1-0.3 M NaCl) exhibits nucleosomal periodicity and is found in particles which have the same electrophoretic mobility as bona fide H1- or high mobility group protein-containing mononucleosomes. Thus, factors believed to be involved with both the higher order coiling and transcriptionally active state of chromatin are rapidly complexed with newly synthesized DNA and may be retained on parental nucleosomes throughout replication. In contrast, approximately 40% of new DNA is resistant to extraction with solutions of moderate ionic strength. Most of this material is eluted from nuclei by 0.4-0.6 M NaCl. While bulk chromatin that is extracted by 0.4-0.6 M NaCl is organized into nucleosomes, most of the newly replicated "chromatin" from the same fractions lacks subunit structure, as determined by DNA size analyses in polyacrylamide gels, thereby distinguishing this nascent material from newly replicated chromatin eluted at lower ionic strength. Within 15 min all newly synthesized chromatin matures and exhibits the solubility and nucleosomal periodicity characteristics of bulk chromatin. The unusual properties of the "nonnucleosomal" fractions may reflect the structure of newly synthesized DNA prior to its assembly into nucleosomes.

Chromatin↗
PubMed1981

In vivo assembly of newly synthesized histones.

Following a labeling period of 2 min, HeLa histones continue to accumulate in chromatin for 10 min, indicating the presence of a histone pool. During the accumulation period, H2A and H2B enter chromatin immediately, while entry of H3 and H4 is more prolonged. Association of newly synthesized core histones with chromatin does not necessarily indicate assembly. When 2-min [3H]lysine-labeled chromatin is exposed to 0.45 M NaCl, nearly half of the newly synthesized histones are dissociated, while mature core histones are stable. H2A and 70% of H2B are salt stable and remain with newly synthesized polynucleosomes. About 30% of H2B, 50% of H4, and all of H3 are salt labile; thus, both the new nucleosomal core histones and salt-labile new core histones are nonstoichiometric. Pulse-labeled core histones are more trypsin-sensitive than mature histones. When the salt-labile, newly synthesized histones are removed, the remaining proteins have the same trypsin sensitivity as bulk core protein. Examination of the tryptic peptides indicated that the increased trypsin sensitivity was due to complete destruction of the loosely associated core histones which undergo a lag prior to assembly. The altered order of appearance of two peptides in stripped, newly assembled nucleosomes indicates that the conformation in these particles is different from that in mature chromatin.

Carbon Radioisotopes↗
PubMed1981

Rapid turnover of the histone-ubiquitin conjugate, protein A24.

The specific activity of protein A24 was found to exceed that of the core histones by 2-3 fold following a brief labeling period. Accordingly, the A24 protein was found to be unstable, with a decay half-life of 90 minutes. When decay of the ubiquitin moiety was measured, it was found to turn over more extensively than the H2A moiety.

Chromatin↗
PubMed1978

Nucleosomes associated with newly replicated DNA have an altered conformation.

In vitro DNA synthesis was studied in HeLa cell nuclei, with emphasis on the question of whether newly replicated DNA is associated with nucleosomes. The newly replicated DNA was twice as sensitive to digestion by micrococcal nuclease as mature chromatin DNA, reaching a limit digest at 20-25% acid-insoluble product. Examination of the intermediates of digestion by micrococcal nuclease showed the nuclease-resistant, new DNA to be complexed in nucleosomes. However, structural differences were evident at both the polynucleosomal and the core particle level. The nucleosomes on newly replicated DNA were arranged with a repeat size of 165-170 base pairs-i.e., smaller than the 185-base-pair repeat of mature chromatin. The heterogeneity of polynucleosomal multimers, evident in digests of whole chromatin, was reduced in newly replicated chromatin such that the multimers resolved as sharply defined bands. Nucleosomal core particles associated with newly replicated DNA had a different conformation from particles in mature chromatin based on the following lines of evidence: (i) during micrococcal nuclease digestion, the monomer nucleosomes did not accumulate but were rapidly degraded under certain conditions; (ii) micrococcal nuclease limit digest patterns and DNase I digestion patterns, both of which reflect internal nucleosomal protein DNA associations, differed significantly from control patterns. These findings bear directly on models postulated for nucleosome-DNA interactions during chromation replication. A possible mechanism to account for the conformational change and its role in replication are discussed.

Chromatin↗