Characterization of proteins associated with nuclear ribonucleoprotein particles by two-dimensional polyacrylamide gel electrophoresis.

Rat liver nuclear ribonucleoprotein particles were prepared by two different methods and defined as 40S ribonucleoprotein (40S RNP) and heterogeneous nuclear ribonucleoprotein (HnRNP) particles. The RNP particles were either solubilized in 8 M urea--6 mM 2-mercaptoethanol--20 mM glycine--20 mM Tris--HCl (pH 8.4) or subjected to removal of RNA by phenol extraction prior to solubilizing the proteins in the urea buffer. The proteins associated with 40S RNP and HnRNP were heterogeneous and very similar in their electrophoretic patterns when analyzed by two-dimensional PAGE, except a protein with molecular weight of 62 000 and an isoelectric point (pI) of 6.2 was present only in HnRNP particles. At least 12 major and 22 minor components could be identified in both preparations. The major proteins were found at pI values varying from 6.0 to 8.5 and with molecular weights from 32 000 to 42 000, and a group of proteins with molecular weight approximately 65 000 were more prominent in HnRNP than in 40S RNP. The other components were found mainly at pI ranges from 5.0 to 6.5 with molecular weights from 43 000 to 65 000. The phenol method extracted essentially all proteins associated with either 40S RNP and HnRNP, but was less effective in extracting a group of proteins with pI values from 5.0 to 5.5 and more efficient for proteins with pI values from 7.5 to 8.5. When chromatin proteins isolated by phenol extraction were compared with HnRNP particle proteins isolated by the same method, the electrophoretic mobilities of the HnRNP particle proteins were found to be identical with a fraction nonhistone chromatin proteins. The 40S RNP particles were further purified by metrizamide isopycnic density gradient centrifugation. The electrophoretic patterns of these proteins were very similar to those prepared by sucrose density gradient centrifugation. Therefore, we concluded that the proteins of RNP particles constituted part of the chromatin proteins.     

High-mobility group chromosomal proteins of wheat

Four proteins have been extracted from purified chromatin of wheat embryos with 0.35 M NaCl. These proteins are soluble in 2% (w/v) trichloroacetic acid and thus meet the original operational requirements to be classified as "high-mobility group" (HMG) chromosomal proteins. The proteins have been characterized by one- and two-dimensional electrophoresis, amino acid analysis, and peptide mapping. Three of the proteins (HMGb, c, and d) share the mammalian HMG characteristic of being rich in both acidic and basic amino acid residues. Unlike their putative mammalian counterparts, these plant HMG proteins contain less than 7 mol % proline. The fourth wheat protein (HMGa) is rich in both proline and in basic amino acid residues. This wheat protein, however, contains only about half the proportion of acidic residues found in mammalian HMG proteins--a characteristic also found in the trout testis HMG protein, H6. Comparative peptide maps show that none of the wheat HMG proteins are degradation products of other HMG proteins or the H1 histones. The peptide maps have not, however, been useful in establishing homologies with mammalian HMG proteins. Wheat HMG proteins are released from DNase I-treated nuclei and co-isolate with micrococcal nuclease-sensitive chromatin fractions. Similar observations concerning the HMG proteins of vertebrate animals have been considered consistent with a role for these proteins as structural components of actively transcribed chromatin.     

Multiacetylated forms of H4 are found in a putative transcriptionally competent chromatin fraction from trout testis.

We have examined the distribution of acetylated histones derived from various trout testis chromatin fractions of different composition. Our results indicate that a chromatin fraction, preferentially solubilized by micrococcal nuclease, containing the bulk of the HMG proteins and similar to a fraction released from intact trout nuclei and previously shown to be enriched in transcribed DNA sequences also possesses high levels of multiacetylated species of H4. Histones 2A, 2B and 3 are also acetylated in this particular chromatin fraction. Monoacetylated species of the 4 inner nucleosomal histones appear to be characteristic of the nucleohistone portion of trout testis chromatin.     

Protein distribution amongst chromatin particles following DNAse II digestion

When active chromatin is released as a Mg-soluble fraction following digestion of nuclei with DNAse II, as concomitant release of HMG proteins, and hnRNP particles occurs. Release of HMG 14 and 17 is dependent on active chromatin release, whereas HMG 1 and 2, and hnRNP particles are released in an independent process. The Mg-soluble fraction comprises a heterogenous mixture of particles of less compact conformation than normal nucleosomes, and prone to protein-induced aggregation. Histone H1, and HMG 14 and 17 appear to be associated with these particles in a reversible manner, whereas HMG 1 and 2 are unbound.     

Enrichment of transcribed and newly replicated DNA in soluble chromatin released from nuclei by mild micrococcal nuclease digestion

A chromatin fraction solubilized from mouse myeloma nuclei under near-physiological ionic conditions by very mild micrococcal nuclease digestion at 0°C is enriched at least 7-fold in DNA complementary to total myeloma polyadenylated mRNA, and 15-fold in DNA originating near the replication fork (labeled within 30 s). Newly replicated DNA recovered in solubilized chromatin after brief labeling was incorporated mainly into particles sedimenting with, or faster than, mononucleosomes. A rapid decrease in enrichment of newly replicated DNA in readily released, soluble chromatin with increasing labeling times indicated that newly replicated chromatin matured within 90 s to a form that was partitioned similarly to bulk chromatin by this fractionation method. Previous studies showed that chromatin readily solubilized from myeloma nuclei is enriched in high-mobility-group (HMG) and other non-histone proteins, RNA and single-stranded DNA; and depleted in H1 and 5-methylcytosine, relative to bulk chromatin (Jackson, J.B., Pollock, J.M., Jr., and Rill, R.L. (1979) Biochemistry 18, 3739–3748). Mild digestion of chicken erythrocyte nuclei with micrococcal nuclease yielded a soluble chromatin fraction (1–2% of the total DNA) with similar properties. This fraction was enriched at least 6-fold in DNA complementary to chicken globin mRNA, relative to total erythrocyte DNA.     

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).     

Antigenic properties of heterogeneous nuclear ribonucleoprotein particles weakly binding nonhistone proteins and proteins binding single-stranded DNA (SSB proteins) from Ehrlich ascites tumor]

Antigenic properties of the proteins of heterogeneous nuclear ribonucleoprotein particles, (hnRNP), weakly bound nonhistone chromatin proteins (WB(N)P) and single-strand DNA-binding proteins (SSB proteins) from chromatin and extrachromatin fraction of the Ehrlich ascites tumor cells have been comparatively studied. The chromatin and extrachromatin SSB proteins displayed similar mobility in the tube and slab SDS/PAGE, had the same ssDNA-binding capacity and similarly stimulated the replicative synthesis in permeable cells. However, the chromatin SSB proteins contained 1.4 times higher phosphate amount than the extrachromatin ones (3.1 and 2. 2. moles phosphorus per 1 mole protein, respectively). The study of four protein groups with the use of a rabbit antiserum to/against extrachromatin SSB proteins (titer 1:13000 by enzyme immunoassay) showed that the chromatin and the extrachromatin SSB proteins have similar antigenic properties. One fraction of the hnRNP proteins was also reactive with the antiserum, whereas the WB(N)P displayed no cross-reactivity. The specificity of the ferm "SSB proteins" as applied to eukaryotic cells, their affinity with hnRNP proteins and differences from the HMG proteins are discussed.     

Comparative studies on microinjected high-mobility-group chromosomal proteins, HMG1 and HMG2

The nonhistone chromosomal proteins, HMG1 and HMG2, were iodinated and introduced into HeLa cells, bovine fibroblasts, or mouse 3T3 cells by erythrocyte-mediated microinjection. Autoradiographic analysis of injected cells fixed with glutaraldehyde consistently showed both molecules concentrated within nuclei. Fixation with methanol, on the other hand, resulted in some leakage of the microinjected proteins from the nuclei so that more autoradiographic grains appeared over the cytoplasm or outside the cells. Both injected and endogenous HMG1 and HMG2 partitioned unexpectedly upon fractionation of bovine fibroblasts, HeLa, or 3T3 cells, appearing in the cytoplasmic fractions. However, in calf thymus, HMG1 and HMG2 molecules appeared in the 0.35 M NaCl extract of isolated nuclei, as expected. These observations show that the binding of HMG1 and HMG2 to chromatin differs among cell types or that other tissue-specific components can influence their binding. Coinjection of [125I]HMG1 and [131I]HMG2 into HeLa cells revealed that the two molecules display virtually equivalent distributions upon cell fractionation, identical stability, identical intracellular distributions, and equal rates of equilibration between nuclei. In addition, HMG1 and HMG2 did not differ in their partitioning upon fractionation nor in their stability in growing vs. nongrowing 3T3 cells. Thus, we have not detected any significant differences in the intracellular behavior of HMG1 and HMG2 after microinjection into human, bovine, or murine cells.     

Binding of HMG14 non-histone protein to histones H2A, H2B, H1 and DNA in reconstituted chromatin

The interaction between calf thymus HMG14 and rat liver chromatin components has been studied via reconstitution and chemical cross-linking. Selective labeling of HMG14 with photoactivable reversible heterobifunctional reagents has allowed a clear identification of the histones interacting with it (histones H2A, H2B and H1). These results are not dependent on whether the chromatin samples used were bulk chromatin, mononucleosomes, or core particles (for H2A and H2B). In addition to histone proteins, DNA also seems to be involved in HMG14 attachment to nucleosome.     

The distribution of nuclear proteins and transcriptionally-active sequences in rat liver chromatin fractions

Chromatin fractions from rat liver nuclei digested by nucleases were separated by differential solubility into several fractions. Material solubilized during digestion (predominantly monomer nucleosomes and polynucleosomes) had the highest HMG14 + 17/DNA ratios but were not enriched in active gene sequences (albumin and c-Ha-ras1 genes). Material soluble in a low ionic strength buffer containing 0.2 mM MgCl2 (monomer nucleosomes and polynucleosomes) contained in addition to the histones, HMG14 and 17 plus a 41K non-histone protein. This fraction was depleted in active gene sequences and enriched in inactive sequences. The insoluble material was highly enriched in active sequences and had the lowest HMG14 + 17/DNA ratio. This fraction could be further fractionated into a histone-containing 2 M NaCl-soluble fraction and a 2 M NaCl-insoluble matrix-bound fraction, both of which were enriched in active sequences. The results show that the HMG proteins do not partition with active sequences during fractionation of chromatin. The 41K protein may be associated with inactive chromatin fraction.     

Comparison of the high mobility group proteins and their chromatin distribution in trout testis and liver

The trout testis contains two major high mobility group (HMG) proteins HMG-T and H6 which, although related to the four mammalian HMGs, exhibit distinct variation as evidenced by differences in electrophoretic mobility and amino acid sequence. Previous work using various endonucleases as probes has shown that HMG-T and H6 are located at specific sites in the testis chromatin. The differentiation of testis cells during spermatogenesis is characterized by a unique transition from a histone-packaged genome to one bound by a class of small molecular weight, highly basic proteins, the protamines. Questions arise as to whether any of the HMG variability may be unique to the process of spermatogenesis and whether the histone-protamine transition occurring in most testis cells affects the HMG protein distribution and/or the specificity of the probe. In an attempt to answer these questions, the distribution of the HMG proteins in the chromatin of trout liver, a tissue lacking protamine, has been studied and comparisons made with testis. Liver HMGs exhibit the same electrophoretic characteristics as the testis HMGs indicating that the variability when compared to mammalian HMGs is primarily phylogenetic in origin rather than tissue-specific. Furthermore, micrococcal nuclease digestion of liver nuclei and its effect on the subsequent HMG protein distribution during chromatin fractionation yields a pattern very similar to that for testis, suggesting that the interaction of the HMGs with the remaining testis nucleohistone is not significantly altered by the ongoing transition to nucleoprotamine. Finally, the HMGs represent an unusually high proportion of the total testis non-histone protein population; the implications of this are discussed.     

Proteins associated with heterogeneous nuclear RNA in eukaryotic cells

When HeLa cell nuclei axe mechanically disrupted in either hypotonic or isotonic buffers, heterogeneous nuclear RNA is recovered from the post-nucleolar fraction in the form of EDTA-resistant ribonucleoprotein particles, which sediment between 40 S and 250 S in sucrose gradients containing 0.01 m or 0.15 m-NaCl. That the RNA in these particles is HnRNA² is indicated by its heterodisperse sedimentation (20 to 80 S) and its continued synthesis in concentrations of actinomycin D that selectively inhibit the synthesis of ribosomal RNA. The specificity of the HnRNA-protein complexes is evidenced by the failure of deliberate attempts to generate artificial RNP by the addition of deproteinized HnRNA to intact or disrupted nuclei at low ionic strength.The proteins bound to HnRNA are complex. In HeLa cells, HnRNP particles contain proteins with molecular weights from 39,000 to approximately 180,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric points between 4.9 and 8.3 (analytical isoelectric focusing). They are readily distinguishable from proteins in other cell fractions, including those in chromatin.Exposure of HeLa HnRNP particles to 0.5 m-NaCl reduces their average sedimentation velocity by approximately 30%. CsCl density-gradient analysis reveals that this is accompanied by the loss of a major portion of the proteins. However, a significant fraction of the HnRNP (25 to 30%) is resistant to high salt concentrations and continues to band at the same density as native HnRNP (1.43 g/cm³). This is true even after prolonged exposure (24 h) to high salt. The salt-resistant HnRNP is enriched for proteins above 60,000 molecular weight. In at least these two respects, this sub-class of HnRNP resembles “messenger RNP” prepared from cytoplasmic polyribosomes, which is also salt-stable and contains relatively high molecular weight proteins.HnRNP particles can also be recovered from HeLa cell nuclei lysed in high salt but these contain many extra proteins, notably histones, and sediment much faster in sucrose gradients than particles prepared as above. HnRNP is not liberated by extracting HeLa nuclei in 0.14 m-NaCl, pH 8.0 (Samarina et al., 1967) unless the temperature is 20 °C or higher. In this case the particles are converted to 45 S structures, which contain partially degraded HnRNA. 45 S particles can also be produced by subjecting 40 to 250 S HnRNP to a very limited digestion with pancreatic ribonuclease (1 to 2 hits/molecule).HnRNP particles have similar sedimentation velocities (40 to 300 S) when isolated under physiological ionic conditions from a variety of mammalian cells, including WI38 human diploid fibroblasts, mouse L-cells, monkey kidney cells and rat liver. However, electrophoresis reveals a distinct pattern of HnRNP proteins for each cell type. It is proposed that this cell-specificity reflects a situation in which HnRNA molecules that differ in nucleotide sequence are complexed with different sets of proteins, so that the resulting HnRNP particles are biochemically distinct at each genetic locus. This hypothesis is discussed in relation to the cytology of lampbrush and polytene chromosomes.     

The effect of a high mobility group protein (HMG 17) on the structure of acetylated and control core HeLa cell chromatin

The effect of binding a high mobility group protein (HMG 17) on the stability and conformation of acetylated and control HeLa high molecular weight core chromatin (stripped of H1 and non-histone chromosomal proteins) was studied by circular dichroism and thermal-denaturation measurements. Previously it had been shown that conformational differences exist between native whole chromatin derived from butyrate-treated (acetylated) and control HeLa cells and that these conformational differences disappear by removing H1 and non-histone chromosomal proteins (Reczek, P.R., Weissman, D., Huvos, P.E. and Fasman, G.D. (1982) Biochemistry 21, 993–1002). The circular dichroism spectra and the thermal denaturation profiles of control and acetylated core chromatin were found to be similar. The circular dichroism properties of HMG 17 reconstituted highly acetylated and control core chromatin indicated the same alteration of chromatin structure at low ionic strength (1 mM sodium phosphate/0.25 mM EDTA, pH 7.0). The magnitudes of the decrease in ellipticity were proportional to the amount of HMG 17 bound and were found to be the same for both the acetylated and control core chromatin. Thermal denaturation profiles confirmed this change in structure induced by HMG 17 on control and highly acetylated core chromatin. The thermal denaturation profiles, which were resolved into three component transitions, exhibited a shifting of hyperchromicity from the lower melting transitions to the higher melting transitions, with a concomitant rise in T_m, on HMG 17 binding to both control and acetylated chromatin. The natures of the interactions of HMG 17 at higher ionic strength (50 mM NaCl/0.25 mM EDTA/1 mM sodium phosphate, pH 7.0) with acetylated and control core chromatin were slightly different, as measured by circular dichroism; however, a decrease in ellipticity was observed for both samples upon binding of HMG 17. These observations suggest that acetylation coupled with HMG 17 binding to core chromatin does not loosen chromatin structure. HMG 17 binding to control and acetylated core chromatin produces an overall stabilization and compaction of chromatin structure.     

Antibodies to 5 M urea soluble chromosomal proteins from HeLa cells

The tissue specificity of a chromosomal protein fraction, extractable from chromatin with 5 M urea at low ionic strength, has been examined in HeLa, A549 and HT 29 cells. Electrophoresis in polyacrylamide gels indicates that each cell type has a different content of 5 M urea soluble proteins which are distinguishable from the histones, from the tight DNA-binding proteins and from the high-mobility-group chromosomal proteins. Antibodies against 5 M urea soluble proteins extracted from HeLa cells were produced in mice. Although each of the mice tested prior to immunization contained a detectable amount of antibodies against both the 5 M urea soluble proteins and tight DNA-binding proteins, immunization elevated the level of the antibodies in the serum over 100-fold. The antibodies do not distinguish between the 5 M urea extracts obtained from different sources because most of the antibodies are directed against antigens shared by the cells studied. Immunofluorescence studies reveal that components which cross-react with 5 M urea soluble chromosomal proteins are also present in the cytoplasm. We conclude the following. (1) 5 M urea extracts from chromatin a group of proteins which differs among cells. (2) Mice contain detectable amounts of autoantibodies against these chromosomal proteins. (3) Immunization with the 5 M urea extractable fraction elicits antibodies against a restricted number of antigenic components which are shared among the cells studied. (4) 5 M urea extractable proteins are found both in the nucleus and cytoplasm; part of these may be cytoskeletal elements. Because the antisera do not react with histones, high-mobility-group proteins and tight DNA-binding proteins, they may be used for various functional studies on the 5 M urea extractable chromosomal protein fraction.     

Upon the observation of superbeads in chromatin.

There exist some indications that nucleases recognize "superbeads" in chromatin. We show that a chromatin extract of rat liver which contains so-called "superbead"-peaks can be separated in a Mg++ soluble and a Mg++ insoluble fraction. The Mg++ insoluble fraction contains the full complement of histones and the expected DNA fragments, but has lost the characteristic peaks in sucrosegradient profiles. These discrete peaks are found in the Mg2+ soluble fraction of the chromatin extract. We give evidence that these peaks are RNP particles on the basis of their protein- and nucleic acid contents.     

Subcellular location of polypeptides that react with anti-Sm and anti-RNP antibodies

Whole nuclear and cytoplasmic fractions from HeLa cells were analyzed in protein gel blots probed with either monoclonal anti-Sm or polyclonal anti-(U1)RNP antibodies. The cells were fractionated by a nonaqueous procedure, to minimize proteolysis and artifactual leakage of nuclear components to the cytoplasmic fraction. Unexpectedly, more reactive proteins were detected in the nucleus than shown earlier in partially purified small nuclear ribonucleoprotein particles (snRNPs). In addition, reactive polypeptides were now found in the cytoplasm. These results are discussed in reference to the possibility that the nucleus and cytoplasm of adult somatic human cells may have a more complex than anticipated set of populations of polypeptides bearing Sm or RNP antigenic determinants, including some proteins that might not be in snRNP form.