Functional regulation of Galpha16 by protein kinase C.

Recent evidence demonstrates that the alpha subunits of some heterotrimeric GTP-binding proteins (G proteins) are subject to modification by protein kinase C (PKC). For the family of G proteins that activate the phospholipase C/inositol trisphosphate/calcium/PKC pathway, such modification could result in G protein autoregulation. To examine the potential regulation of members of the Galphaq family by PKC phosphorylation, we expressed the thyrotropin-releasing hormone (TRH) receptor in combination with Galphaq, Galpha11, Galpha14, Galpha15, or Galpha16 in Xenopus oocytes and examined the regulation of signaling by PKC activators and inhibitors. For Galpha16 and Galpha15, the two family members of hematopoietic lineage, PKC activators reduce both the magnitude and the time course of TRH-mediated responses; PKC inhibitors have the opposite effect. The PKC-mediated effects are evident in measurements of GTPase activity, suggesting that the regulation is occurring early in the signaling pathway. In vivo phosphorylation experiments demonstrate that Galpha16 is a substrate for PKC modification. By comparison, Galphaq is not phosphorylated by PKC in vivo, and oocytes expressing Galphaq are not functionally modulated by PKC. Repeated TRH stimulation of oocytes expressing Galpha16 mimics the effects of PKC activators, and this functional regulation is correlated with an increase in Galpha16 phosphorylation. A mutant Galpha16 with four consensus PKC phosphorylation sites removed is not phosphorylated in vivo, and TRH responses mediated through the mutant are not regulated by PKC. These results demonstrate that signaling involving hematopoietic G proteins is subject to PKC-mediated autoregulation, at least in part, by phosphorylation of the G protein alpha subunit.

G protein-coupled receptor kinase 2 (GRK2): mechanisms of regulation and physiological functions.

G protein-coupled receptor kinase 2 (GRK2) plays a key role in determining the rate and extent of G protein-coupled receptor (GPCR) desensitization and resensitization. Recent data indicate that GRK2 activity, subcellular distribution and expression are tightly regulated. The important physiological function of GRK2 as a modulator of the efficacy of GPCR signal transduction systems is exemplified by its relevance in cardiovascular physiopathology as well as by its emerging role in the regulation of chemokine receptors.

The chemokine monocyte chemotactic protein 1 triggers Janus kinase 2 activation and tyrosine phosphorylation of the CCR2B receptor.

The chemokines are a growing family of low m.w., 70- to 80-residue proinflammatory cytokines that operate by interacting with G protein-coupled receptors. Chemokines are involved in cell migration and in the activation of specific leukocyte subsets. Using the Mono Mac 1 monocytic cell line, we show that monocyte chemotactic protein 1 (MCP-1) triggers activation of the Janus kinase 2 (JAK2)/STAT3 pathway and CCR2 receptor tyrosine phosphorylation. Both Ca2+ mobilization and cell migration are blocked in Mono Mac 1 cells by tyrphostin B42, a specific JAK2 kinase inhibitor. Within seconds of MCP-1 activation, JAK2 phosphorylates CCR2 at the Tyr139 position and promotes JAK2/STAT3 complex association to the receptor. This MCP-1-initiated phosphorylation and association to JAK2 is also observed in CCR2B-transfected HEK293 cells. In contrast, when a CCR2B Tyr139Phe mutant is expressed in HEK293 cells, it is not phosphorylated in tyrosine and triggers neither JAK2/STAT3 activation nor Ca2+ mobilization in response to MCP-1. These results implicate the tyrosine kinase pathway in early chemokine signaling, suggesting a key role for this kinase in later events.

Monocyte chemoattractant protein-1-induced CCR2B receptor desensitization mediated by the G protein-coupled receptor kinase 2.

Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine cytokine family, whose physiological function is mediated by binding to the CCR2 and CCR4 receptors, which are members of the G protein-coupled receptor family. MCP-1 plays a critical role in both activation and migration of leukocytes. Rapid chemokine receptor desensitization is very likely essential for accurate chemotaxis. In this report, we show that MCP-1 binding to the CCR2 receptor in Mono Mac 1 cells promotes the rapid desensitization of MCP-1-induced calcium flux responses. This desensitization correlates with the Ser/Thr phosphorylation of the receptor and with the transient translocation of the G protein-coupled receptor kinase 2 (GRK2, also called beta-adrenergic kinase 1 or betaARK1) to the membrane. We also demonstrate that GRK2 and the uncoupling protein beta-arrestin associate with the receptor, forming a macromolecular complex shortly after MCP-1 binding. Calcium flux responses to MCP-1 in HEK293 cells expressing the CCR2B receptor were also markedly reduced upon cotransfection with GRK2 or the homologous kinase GRK3. Nevertheless, expression of the GRK2 dominant-negative mutant betaARK-K220R did not affect the initial calcium response, but favored receptor response to a subsequent challenge by agonists. The modulation of the CCR2B receptor by GRK2 suggests an important role for this kinase in the regulation of monocyte and lymphocyte response to chemokines.

Desensitization of inositol 1,4,5-trisphosphate/Ca2+-induced Cl- currents by prolonged activation of G proteins in Xenopus oocytes.

Expression of G protein alpha subunits of the Gq family with various G protein-coupled receptors induces activation of an inositol 1,4, 5-trisphosphate (IP3)/Ca2+-mediated Cl- conductance in Xenopus oocytes. Our present data show that two members of this family, the human Galpha16 subunit and the murine homologue Galpha15, can induce both activation and inhibition of these agonist-induced currents. Although extremely low amounts (10-50 pg) of injected Galpha16 subunit cRNA cause modest ( approximately 2-fold) enhancement of ligand-induced Cl- currents in oocytes co-injected with thyrotropin-releasing hormone (TRH) receptor cRNA 48 h postinjection, larger Galpha16 and Galpha15 cRNA injections cause >10-fold inhibition of TRH or 5HT2c receptor responses. The inhibition is analyzed in this study. The inhibited currents are recovered if various Gbetagamma subunit combinations are also expressed with the Galpha subunits. The constitutively active mutant, Galpha16Q212L, also causes a strong attenuation of the ligand-induced Cl- currents, but this inhibition is not recovered by co-expression of Gbetagamma subunits. These results indicate that the free Galpha subunit is responsible for the inhibitory signal. Although expression of TRH receptor alone produces maximum responses approximately 48 h after injection, co-expression of TRH receptor with Galpha16 results in enhanced responses 6-12 h postinjection, followed by complete attenuation at 36 h. Furthermore, injection of Galpha16 cRNA alone at comparable levels gives rise to spontaneous Cl- currents within 6-12 h postinjection, suggesting that the early spontaneous activation underlies the later suppression. Expression of other G protein alpha subunits of the Gq family, at cRNA levels considerably higher than effective for Galpha16, produces both analogous spontaneous Cl- currents and, later, inhibition of ligand-induced Cl- currents. Experiments with direct injection of IP3 and of Ca2+ suggest that this inhibition is consistent with the down-regulation of IP3 receptors. These data indicate that both enhancement and inhibition of signaling through G protein-coupled receptors can be mediated by the expression level and/or activity of an individual G protein.

Coupling of the thrombin receptor to G12 may account for selective effects of thrombin on gene expression and DNA synthesis in 1321N1 astrocytoma cells.

In 1321N1 astrocytoma cells, thrombin, but not carbachol, induces AP-1-mediated gene expression and DNA synthesis. To understand the divergent effects of these G protein-coupled receptor agonists on cellular responses, we examined Gq-dependent signaling events induced by thrombin receptor and muscarinic acetylcholine receptor stimulation. Thrombin and carbachol induce comparable changes in phosphoinositide and phosphatidylcholine hydrolysis, mobilization of intracellular Ca2+, diglyceride generation, and redistribution of protein kinase C; thus, activation of these Gq-signaling pathways appears to be insufficient for gene expression and mitogenesis. Thrombin increases Ras and mitogen-activated protein kinase activation to a greater extent than carbachol in 1321N1 cells. The effects of thrombin are not mediated through Gi, since ribosylation of Gi/Go proteins by pertussis toxin does not prevent thrombin-induced gene expression or thrombin-stimulated DNA synthesis. We recently reported that the pertussis toxin-insensitive G12 protein is required for thrombin-induced DNA synthesis. We demonstrate here, using transfection of receptors and G proteins in COS-7 cells, that G alpha 12 selectively couples the thrombin receptor to AP-1-mediated gene expression. This does not appear to result from increased mitogen-activated protein kinase activity but may reflect activation of a tyrosine kinase pathway. We suggest that preferential coupling of the thrombin receptor to G12 accounts for the selective ability of thrombin to stimulate Ras, mitogen-activated protein kinase, gene expression, and mitogenesis in 1321N1 cells.

A novel form of the G protein beta subunit Gbeta5 is specifically expressed in the vertebrate retina.

The G protein beta subunit, Gbeta5, is predominantly expressed in the central nervous system. In rodent brain, Gbeta5 is expressed as a protein with an apparent molecular mass of 39,000 daltons (39 kDa). We have identified an additional Gbeta5 immunoreactive protein of apparent size 44 kDa in the vertebrate retina. Molecular cloning and sequencing of polymerase chain reaction products revealed that the cDNA encoding the larger species of Gbeta5 (Gbeta5L) was identical to the shorter form with the addition of 126 base pairs of 5' DNA sequence potentially encoding an in-frame 42-amino acid extension. Sequencing of mouse Gbeta5 genomic clones demonstrated that the 126-base pair of retinal-specific coding material is derived from a hitherto undetected 5' exon. During sucrose density gradient fractionation of bovine retinas, the 44-kDa Gbeta5L protein co-purified with rod outer segment membranes. Incubation of rod outer segment membranes with the nonhydrolyzable guanine nucleotide, GTPgammaS (guanosine 5'-3-O-(thio)triphosphate), which released the Gbeta subunit of transducin (Gbeta1), failed to remove Gbeta5L. The 39-kDa Gbeta5 protein displayed differential association with retinal and brain membranes. In the retina, Gbeta5 was present as a soluble protein and was undetectable in the membrane fraction, whereas in the brain approximately 70% of Gbeta5 was associated with cellular membranes. In transient COS-7 cell expression experiments, Gbeta5L formed functional Gbetagamma dimers and Galphabetagamma heterotrimers, and activated phosphoinositide-specific phospholipase Cbeta2 in a manner indistinguishable from the 39-kDa Gbeta5 protein. The cloning of the retinal-specific Gbeta5L cDNA suggests the existence of potentially novel G protein-mediated signaling cascades in photoreception.

Cell-specific signal transduction of parathyroid hormone (PTH)-related protein through stably expressed recombinant PTH/PTHrP receptors in vascular smooth muscle cells.

PTH-related protein activates a G protein-coupled PTH/PTHrP receptor in many cell types and produces diverse biological actions. To study the signal transduction events associated with biological activity of the PTH/PTHrP receptor in vascular smooth muscle, a principal PTHrP-responsive tissue, rat aortic smooth muscle cells (A10) were stably transfected with a plasmid encoding a PTH/PTHrP receptor and tested for ligand binding, PTHrP-(1-34)-induced cAMP levels, inositol phosphate production, and cytosolic calcium transients. Of nineteen G418-resistant lines recovered, all exhibited high affinity binding [approximately dissociation constant (Kd) > 10(-10)) of iodinated [Tyr36]hPTHrP(1-36)NH2 and ligand-induced cAMP accumulation (2- to 100-fold), which was directly proportional to PTH/PTHrP receptor number (range 4 x 10(3) to 7 x 10(7) sites/cell]. PTHrP had no effect on intracellular calcium or inositol phosphate formation in any cell line regardless of receptor number despite the presence of detectable G alpha q). Transient overexpression of individual G alpha q proteins (G alpha q, G alpha 11 or G alpha 14) into PTH/PTHrP receptor-expressing A10 cells conferred the ability of PTHrP to increase intracellular calcium and inositol phosphate formation. Ligand activation of the recombinant PTH/PTHrP receptor elicited appropriate downstream biological effects in A10 cells including inhibition of DNA synthesis and osteopontin messenger RNA (mRNA) expression. Thus, a single PTH/PTHrP receptor, though capable of coupling to different G proteins, signals exclusively through a cAMP-dependent pathway in vascular smooth muscle.

Increased expression of alphaq family G-proteins during oocyte maturation and early development of Xenopus laevis.

G-proteins of the alphaq family link extracellular stimulation of plasma membrane receptors to phospholipase C and consequently to intracellular Ca2+ release. Because they might function in initiating Ca2+ release at fertilization, we examined Galphaq family proteins in oocytes and eggs of Xenopus laevis. Three members of this protein family were identified by immunoblotting and antisense depletion. These proteins are barely detectable in the immature oocyte, but undergo a 6-fold increase in amount during oocyte maturation. This increase in Galphaq family protein expression correlates with the acquisition, during oocyte maturation, of the ability to release Ca2+ at fertilization (Schlichter and Elinson, 1981, Dev. Biol. 83, 33-41). In contrast, amounts of Galphas and Galphai3 are constant during maturation. We also examined the amounts of Galphaq, Galphas, and Galphai3 proteins during early development. While amounts of Galphas and Galphai3 show little or no change, Galphaq family protein expression increases 27-fold between the egg and neurula stages, suggesting that these proteins may be important in initiating Ca2+ release during early development.

G12 requirement for thrombin-stimulated gene expression and DNA synthesis in 1321N1 astrocytoma cells.

Thrombin stimulation of 1321N1 astrocytoma cells leads to Ras-dependent AP-1-mediated transcriptional activation and to DNA replication. In contrast to what has been observed in most cell systems, in 1321N1 cells these responses are pertussis toxin-insensitive. The pertussis toxin-insensitive G-protein G12 has been implicated in cell growth and transformation in different cell systems. We have examined the potential role of this protein in AP-1-mediated transcriptional activation and DNA synthesis in 1321N1 cells. Transient expression of an activated (GTPase-deficient) mutant of G alpha 12 increased AP-1-dependent gene expression. This response was inhibited by co-expression of a dominant negative Ala-15 Ras protein. To determine whether the pertussis toxin-insensitive G12 protein is involved in the thrombin-stimulated DNA synthesis, an inhibitory antibody against the C-terminal sequence of G alpha 12 subunit was microinjected into 1321N1 cells. Microinjection of the anti-G alpha 12 resulted in a concentration-dependent inhibition of thrombin-stimulated DNA synthesis. In contrast, microinjection of nonimmune IgG or an antibody directed against the C terminus of G alpha o did not reduce the mitogenic response to thrombin. Furthermore, microinjection of the anti-G alpha 12 antibody had no effect on fibroblast growth factor-stimulated DNA synthesis. These results demonstrate a specific role for G alpha 12 in the mitogenic response to thrombin in human astroglial cells.

Differential coupling of G protein alpha subunits to seven-helix receptors expressed in Xenopus oocytes.

Xenopus oocytes were used to examine the coupling of the serotonin 1c (5HT1c) and thyrotropin-releasing hormone (TRH) receptors to both endogenous and heterologously expressed G protein alpha subunits. Expression of either G protein-coupled receptor resulted in agonist-induced, Ca(2+)-activated Cl- currents that were measured using a two-electrode voltage clamp. 5HT-induced Cl- currents were reduced 80% by incubating the injected oocytes with pertussis toxin (PTX) and inhibited 50-65% by injection of antisense oligonucleotides to the PTX-sensitive Go alpha subunit. TRH-induced Cl- currents were reduced only 20% by PTX treatment but were inhibited 60% by injection of antisense oligonucleotides to the PTX-insensitive Gq alpha subunit. Injection of antisense oligonucleotides to a novel Xenopus phospholipase C-beta inhibited the 5HT1c (and Go)-induced Cl- current with little effect on the TRH (and Gq)-induced current. These results suggest that receptor-activated Go and Gq interact with different effectors, most likely different isoforms of phospholipase C-beta. Co-expression of each receptor with seven different mammalian G protein alpha subunit cRNAs (Goa, Gob, Gq, G11, Gs, Golf, and Gt) was also examined. Co-expression of either receptor with the first four of these G alpha subunits resulted in a maximum 4-6-fold increase in Cl- currents; the increase depended on the amount of G alpha subunit cRNA injected. This increase was blocked by PTX for G alpha oa and G alpha ob co-expression but not for G alpha q or G alpha 11 co-expression. Co-expression of either receptor with Gs, Golf, or Gt had no effect on Ca(2+)-activated Cl- currents; furthermore, co-expression with Gs or Golf also failed to reveal 5HT- or TRH-induced changes in adenylyl cyclase as assessed by activation of the cystic fibrosis transmembrane conductance regulator Cl- channel. These results indicate that in oocytes, the 5HT1c and TRH receptors do the following: 1) preferentially couple to PTX-sensitive (Go) and PTX-insensitive (Gq) G proteins and that these G proteins act on different effectors, 2) couple within the same cell type to several different heterologously expressed G protein alpha subunits to activate the oocyte's endogenous Cl- current, and 3) fail to couple to G protein alpha subunits that activate cAMP or phosphodiesterase.

Narrow A/T-rich zones present at the distal 5'-flanking sequences of the zein genes Zc1 and Zc2 bind a unique 30 kDa HMG-like protein.

Nuclear extracts from maize endosperm were used to investigate protein-DNA interactions in the 5'-upstream region of the Zc1 and Zc2 genes. These genes encode for zeins of apparent molecular mass (MWapp) 16 and 28 kDa, respectively, which accumulate in the endosperm during seed maturation. Binding assays revealed specific binding of a nuclear protein to three A/T-rich elements, 0.9-1.0 kbp upstream from the initiation codon. One of these elements (41 bp, 88% A/T), present in Zc1, contained a 13 nucleotide duplication. The other two (28 bp, 86% A/T; 42 bp alternating A-T) are consecutive elements in Zc2. Competition experiments strongly suggest that the three elements bind to the same protein. Protein-DNA interaction was detected in endosperm nuclear extracts of 8 to 21 days after pollination (DAP), as well as in 25 DAP embryos and in different tissues from plantlets. The protein factor has an MWapp of ca. 30 kDa. This factor has properties suggesting it is an HMG-like protein. These results are consistent with a growing accumulation of data for a number of genes indicating that A/T-rich elements, located at distal and proximal zones of the 5'-flanking sequences, interact with HMG-like proteins.

Random mutagenesis of G protein alpha subunit G(o)alpha. Mutations altering nucleotide binding.

Nucleotide binding properties of the G protein alpha subunit G(o)alpha were probed by mutational analysis in recombinant Escherichia coli. Thousands of random mutations generated by polymerase chain reaction were screened by in situ [35S]GTP gamma S (guanosine 5'-(3-O-thio)-triphosphate) binding on the colony lifts following transformation of bacteria with modified G(o)alpha cDNA. Clones that did not bind the nucleotide under these conditions were characterized by DNA sequence analysis, and the nucleotide binding properties were further studied in crude bacterial extracts. A number of novel mutations reducing the affinity of G(o)alpha for GTP gamma S or Mg2+ were identified. Some of the mutations substitute amino acid residues homologous to those known to interact with guanine nucleotides in p21ras proteins. Other mutations show that previously unstudied residues also participate in the nucleotide binding. Several mutants lost GTP gamma S binding but retained the capacity to interact with the beta gamma subunit complex as determined by pertussis toxin-mediated ADP-ribosylation. One of these, mutant S47C, was functionally expressed in Xenopus laevis oocytes along with the G protein-coupled thyrotropin-releasing hormone (TRH) receptor. Whereas wild-type G(o)alpha increased TRH-promoted chloride currents, S47C significantly decreased the hormone-induced Cl- response, suggesting that this mutation resulted in a dominant negative phenotype.

The G alpha q and G alpha 11 proteins couple the thyrotropin-releasing hormone receptor to phospholipase C in GH3 rat pituitary cells.

Thyrotropin-releasing hormone stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in GH3 cell membranes. The stimulation of the phosphoinositide phospholipase C (PI/PLC) activity can be blocked by incubation of GH3 membranes with polyclonal antibodies directed against a peptide derived from the C-terminal region of G alpha q and G alpha 11. Antibodies directed against the C-terminal region of other G alpha-subunits had no detectable effect. The inhibition was specific since addition of the peptide that was used to prepare the antibody completely reversed the inhibition. Further evidence for the coupling of the TRH receptor to G alpha q or G alpha 11 comes from a reconstitution experiment in which human embryonic kidney cells were transiently transfected with cDNAs corresponding to the TRH receptor, G alpha q or G alpha 11. The PIP2 hydrolysis detected with membranes from cells that over-expressed the TRH receptor alone was low, however, co-expression with the G alpha q or G alpha 11 subunits produced a synergistic stimulation of PI-PLC activity. In contrast, co-expression of these alpha-subunits with the M2 muscarinic acetylcholine receptor induced a weak stimulation of PIP2 hydrolysis. The results presented here suggest that the TRH-dependent stimulation of PI-PLC in GH3 cells is mediated through the G-protein alpha-subunits, G alpha q and/or G alpha 11.

Different mechanism for in vitro formation of nucleosome core particles.

The interaction of different histone oligomers with nucleosomes has been investigated by using nondenaturing gel electrophoresis. In the presence of 0.2 M NaCl, the addition of the pairs H2A,H2B or H3,H4 or the four core histones to nucleosome core particles produces a decrease in the intensity of the core particle band and the appearance of aggregated material at the top of the gel, indicating that all these histone oligomers are able to associate with nucleosomes. Equivalent results were obtained by using oligonucleosome core particles. Additional electrophoretic results, together with second-dimension analysis of histone composition and fluorescence and solubility studies, indicate that H2A,H2B, H3,H4, and the four core histones can migrate spontaneously from the aggregated nucleosomes containing excess histones to free core DNA. In all cases the estimated yield of histone transfer is very high. Furthermore, the results obtained from electron microscopy, solubility, and supercoiling assays demonstrate the transfer of excess histones from oligonucleosomes to free circular DNA. However, the extent of solubilization obtained in this case is lower than that observed with core DNA as histone acceptor. Our results demonstrate that nucleosome core particles can be formed in 0.2 M NaCl by the following mechanisms: (1) transfer of excess core histones from oligonucleosomes of free DNA, (2) transfer to excess H2A,H2B and H3,H4 associated separately with oligonucleosomes to free DNA, (3) transfer to excess H2A,H2B initially associated with oligonucleosomes to DNA, followed by the reaction of the resulting DNA-(H2A,H2B) complex with oligonucleosomes containing excess H3,H4, and (4) a two-step transfer reaction similar to that indicated in (3), in which excess histones H3,H4 are transferred to DNA before the reaction with oligonucleosomes containing excess H2A,H2B. The possible biological implications of these spontaneous reactions are discussed in the context of the present knowledge of the nucleosome function.

The b-32 protein from maize endosperm: characterization of genomic sequences encoding two alternative central domains.

As derived from a cDNA clone, the structure of the b-32 protein of Zea mays, a putative regulatory factor of zein expression, has a central acidic region separated by two domains covered by secondary structure motifs. In this work, three b-32 genomic clones were selected from two genomic libraries obtained from the maize inbred lines W64A and A69Y. The nucleotide sequences of the complete coding region of each b-32 gene, as well as long stretches of their 5' and 3' flanking regions, were determined. Introns are not present in the b-32 genomic sequences. Minor variations among the three genes and an earlier reported b-32 cDNA indicates that they constitute a gene family showing a characteristic polymorphism. Such a polymorphism is highly evident in large segments of the upstream regulatory sequences. Interestingly, when compared with cDNA (W64A) or with gene b-32.120 (W64A), the genes b-32.129 (W64A) and b-32.152 (A69Y) show three jumps of the reading frame in the central part of the coding region, resulting in a completely different sequence of the b-32 protein central domain. In all cases, variations in the N- and C-terminal domains account only for microheterogeneity.

Association of nucleosome core particle DNA with different histone oligomers. Transfer of histones between DNA-(H2A,H2B) and DNA-(H3,H4) complexes.

In non-denaturing low ionic strength gels, the titration of core DNA with H2A,H2B produces five well-defined bands. Quantitative densitometry and cross-linking experiments indicate that these bands are due to the successive binding of H2A,H2B dimers to core DNA. Only two bands are obtained with DNA-(H3,H4) samples. The slower of these bands is broad and presumably corresponds to two complexes containing one and two H3,H4 tetramers, respectively. In gels of higher ionic strength, DNA-(H2A,H2B) samples produce an ill-defined band, suggesting that the lifetime of the complexes containing H2A,H2B is relatively short. However, the low intensity of the free DNA band observed in these gels indicates that most of the DNA is associated with H2A,H2B. In agreement with this, our results obtained using different techniques (sedimentation, cross-linking, trypsin and nuclease digestions, and thermal denaturation) demonstrate that the association of H2A,H2B with core DNA occurs in free solution in both the absence and presence of NaCl (0.1 to 0.2 M). The low mobilities of DNA-(H2A,H2B) complexes, together with sedimentation and DNase I digestion results, indicate that the DNA in these complexes is not folded into the compact structure found in the core particle. Furthermore, non-denaturing gels have been used to study the dynamic properties of DNA-(H2A,H2B) and DNA-(H3,H4) complexes in 0.2 M-NaCl. Our results show that: (1) H2A,H2B and H3,H4 can associate, respectively, with DNA-(H3,H4) and DNA-(H2A,H2B) to produce complexes containing the four core histones; (2) DNA-(H2A,H2B) and DNA-(H3,H4) are able to transfer histones to free core DNA; (3) an exchange of histone pairs takes place between DNA-(H2A,H2B) and DNA-(H3,H4) and produces complexes with the same histone composition as that of the normal nucleosome core particle; and (4) although both histone pairs can exchange, histones H2A,H2B show a higher tendency than H3,H4 to migrate from one incomplete core particle to another. The complexes produced in these reactions have the same compact structure as reconstituted core particles containing the four core histones. Our kinetic results are consistent with a reaction mechanism in which the transfer of histones involves direct contacts between the reacting complexes. The possible participation of these spontaneous reactions on the mechanism of nucleosome assembly is discussed.

Fluorescent properties of histone-1-anilinonaphthalene 8-sulfonate complexes in the presence of denaturant agents: application to the rapid staining of histones in urea and Triton-urea-polyacrylamide gels.

In the present report it is shown that histone bands in urea-acetic acid or Triton-urea-acetic acid-polyacrylamide gels can be stained with the fluorescent dye 1-anilinonaphthalene 8-sulfonate and visualized by transillumination of the gel with an uv-light source. The 1-anilinonaphthalene 8-sulfonate staining method described here for urea and Triton-urea gels is rapid (it can be completed in 90 min) and allows the detection of less than 1 micrograms of histone per band.

Rapid fluorescent staining of histones in sodium dodecyl sulfate-polyacrylamide gels.

The increase in the fluorescence intensity of 1-anilinonaphthalene-8-sulfonate (ANS) produced by core histones is higher than that produced by very lysine-rich histones (H1 and H5). In the presence of the anionic detergent sodium dodecyl sulfate (SDS) the enhancement of ANS fluorescence caused by these two groups of histones is roughly the same, but much lower than that observed for core histones in the absence of this detergent. However, the increase of ANS fluorescence produced by histone-SDS complexes is high enough to use it for the staining of these proteins separated in SDS-polyacrylamide gels. Histone bands are stained with ANS after electrophoresis and visualized by transillumination of the gel with a uv light source. The method described in this work allows the rapid detection of less than 0.5 microgram of histone per band.