Further characterization of the posttranslational modifications of core histones in response to heat and arsenite stress in Drosophila

The effects of a heat shock or arsenite treatment on the methylation and acetylation of core histones have been investigated in Drosophila cultured cells. The decrease in H3 methylation, which is observed during a heat shock, is not a demethylation process, but results from methylation arrest. Two-dimensional gel electrophoresis leaves no ambiguity concerning the identity of H2B as a methylated protein, since H2B and D2, a nuclear nonhistone protein, which comigrate on one-dimensional gels, are well separated on these gels. Two-dimensional gel electrophoresis in the presence of Triton X-100 resolves each of the core histones into multiple forms resulting from posttranslational modifications. There are apparently, however, no histone variants in cultured Drosophila cells. At 23 degrees C, the various forms of the core histones resolved on two-dimensional gels are methylated. Under heat-shock or arsenite treatment, the methylation of all forms of H3 is decreased, while that of the various forms of H2B increase. These stress conditions also induce a generalized diminution in the acetylation of all forms of core histones. In the course of a heat shock, the synthesis of H2B is increased and this newly synthesized histone remains unacetylated during the shock. These changes in the patterns of core histone methylation and acetylation may be correlated with the reorganization of gene activity brought about by the heat shock.     

Differential expression of histone sequences in Drosophila following heat shock

The expression of the sequences encoding the four nucleosomal histone proteins was examined following heat shock of a variety of Drosophila cells and was found to be highly differential. In Drosophila melanogaster KC-O cells grown in suspension culture, there is a continuation of the synthesis of all four of the nucleosomal histone proteins following heat shock. Analysis of RNA from these cells confirms that histone messengers are transcribed and located on polysomes. This exact same pattern of histone protein synthesis occurs in KC-O cells grown to low density on plates. In contrast, KC-O cells grown to high density on plates exhibit a dramatic elevation of H2b protein synthesis relative to the synthesis of the other core histones. Organs from D melanogaster third instar larvae were examined to ascertain whether histone protein synthesis continues following heat shock in the organism. Different tissue types exhibited differential histone synthesis. Imaginal disks excised from heat-shocked larvae continue to synthesize nucleosomal histones in a variable fashion. In contrast, neither fat bodies, brains, nor salivary glands continues to synthesize core histone proteins at a significant level. D hydei plated cell cultures and larval tissues fail to synthesize histones at any detectable level following a heat shock. Based on these observations, we propose that there is a differential synthesis of nucleosomal proteins in Drosophila that is highly dependent on the state of the cells prior to the heat shock.     

Patterns of protein synthesis in various cells after extreme heat shock

The analysis of proteins synthesized in rat thymocytes and mouse teratocarcinoma PCC-4 Aza 1 and myeloma Sp2/0 cells after 1 h of treatment at 42 or 44 degrees C was carried out. Shock at 42 degrees C reduced the total synthetic rate of proteins in all three cell lines and induced "classical" heat-shock protein with a mass of 70 kDa (hsp 70). Heat shock at 44 degrees C resulted in almost complete inhibition of protein synthesis; only a small amount of hsp 70 was synthesized. Meanwhile a new 48-kDa polypeptide (pI = 7.5) was found in the cells exposed to severe heat shock. This protein was compared by peptide mapping with other known polypeptides of the same size: heat-shock protein from chicken embryo cells and mitogen-stimulated polypeptide from human lymphoid cells. The peptide maps were not identical. It was also shown that after a shock at 44 degrees C teratocarcinoma cells were able to accumulate anomalous amounts of hsp 70 despite hsp 70 synthesis inhibition. The data show that reaction of various cells to extreme heat shock depends heavily on cell type.     

Histones of Neurospora crassa.

Neurospora crassa chromatin isolated by a rapid method minimizing proteolytic degradation contains approximately one weight of acid-extractable basic protein per weight of DNA. This basic protein consists of five major polypeptide species which are similar in size to the histone proteins of higher eukaryotes and are present in approximately the same molar ratios. These five polypeptides have been purified by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Their electrophoretic mobilities in polyacrylamide gels and their amino acid compositions indicate that they are histones homologous, although not identical, to the H1, H2A, H2B, H3, and H4 histones of mammals. The first 3 residues in the amino acid sequence of Neurospora H3 histone are identical to the first 3 residues in calf and pea H3; Neurospora H1, H2A, and H4 histones have blocked NH2 termini, like their mammalian counterparts. The finding of recognizable H1, H2A, H2B, H3, and H4 histones in Neurospora extends the range of eukaryotes now shown to contain a full complement of these strongly conserved chromosomal proteins, and supports the view that histones became involved in chromosome structure at a very early point in the evolution of eukaryotes.     

Tissue-specific variations in the induction of Hsp70 and Hsp64 by heat shock in insects

The patterns of heat-induced synthesis (37 degrees C to 45 degrees C) of heat shock proteins (Hsps) in different tissues of grasshoppers and cockroaches from natural populations and in laboratory-reared gram-pest (Heliothis armigera) were examined by 35S-methionine labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography. Whereas 45 degrees C was lethal in most cases, optimal induction of Hsp synthesis was seen between 37 degrees C and 42 degrees C. The ongoing protein synthesis was not much affected at these temperatures, except in the tissues of adult H. armigera exposed to 42 degrees C. The profiles of the Hsps induced in the tissues of the insects, however, were different. From the relative abundance of the synthesis of 70-kDa (Hsp70) and 64-kDa (Hsp64) polypeptides, three categories of heat shock response were identified: (1) induction of abundant Hsp70 but little Hsp64 (malpighian tubules, male accessory glands, and ovaries of adult grasshoppers), (2) abundant Hsp64 but little Hsp70 (testes of adult grasshoppers, testes and malpighian tubules of adult cockroaches, and testes, malpighian tubules, and fat bodies of H. armigera larvae), and (3) induction of both Hsp70 and Hsp64 in more or less equal abundance (ovaries of adult cockroaches, salivary glands of H. armigera larvae, and malpighian tubules, male accessory glands, testes, and ovaries of adult H. armigera). Cockroaches collected from storerooms showed detectable synthesis of Hsp64 and/or Hsp70 only after heat shock, but those collected from drains showed detectable synthesis of both Hsp70 and Hsp64 in different tissues without heat stress. Western blotting showed that the 64-kDa polypeptide in these insects is a member of the Hsp60 family. Grasshopper testes, which synthesized negligible Hsp70 but abundant Hsp64 after heat shock, developed thermotolerance. Thus, heat shock response is modulated by developmental and environmental factors in different tissues of insects.     

Acetylation and methylation patterns of core histones are modified after heat or arsenite treatment of Drosophila tissue culture cells

Exposure of Drosophilamelanogaster tissue culture cells to 37 degrees C (heat shock) or to arsenite induces a severe deacetylation of core histones and blocks the methylation of histone H(3) and H(4). Heat shock induces the methylation of histone H(2b). These results are discussed in view of chromatin structure and function.Images     

Inhibition of HeLa cell protein synthesis under heat shock conditions in the absence of initiation factor eIF-2 alpha phosphorylation

Subjecting a HeLa cell suspension culture to an increase in incubation temperature (from 37 degrees to 42 degrees C) results in the rapid cessation of polypeptide chain synthesis followed by a gradual increase in the synthesis of a class of polypeptides referred to as the heat-shock proteins. It has been proposed that the initial, rapid shutoff of protein synthesis (less than 20 min) is due to the phosphorylation of initiation factor eIF-2 in its alpha subunit, a modification known to result in the inhibition of polypeptide synthesis. Using an in vitro translation system derived from heat-shocked HeLa cells grown in suspension culture, we were unable to find any evidence implicating eIF-2 alpha phosphorylation in the initial shutoff of translation during the heat shock response. These results suggest that the rapid inhibition of protein synthesis observed under heat shock conditions is mediated by a mechanism(s) other than eIF-2 alpha phosphorylation.     

Tissue specificity of the heat-shock response in maize

The tissue specificity of the heat-shock response in maize was investigated. The ability to synthesize heat shock proteins (hsp) at 40°C, as well as the intensity and duration of that synthesis, was analyzed in coleoptiles, scutella, green and etiolated leaves, suspension-cultured cells, germinating pollen grains, and primary root sections at different stages of development. One-dimensional sodium dodecyl sulfate gel electrophoresis of extracted proteins revealed that most of the tissues synthesized the typical set of 10 hsp, but that the exact characteristics of the response depended upon the tissue type. While elongating portions of the primary root exhibited a strong heat shock response, the more mature portions showed a reduced ability to synthesize hsp. Leaves, whether green or etiolated, excised or intact, constitutively synthesized a low level of hsp at 25°C, and high levels could be induced at 40°C. Suspension-cultures of Black Mexican sweet corn synthesized, besides the typical set of hsp, two additional polypeptides. In contrast to all the other tissues, germinating pollen grains could not be induced to synthesize the typical set of hsp but did synthesize two new polypeptides of 92 and 56 kD molecular weight.

The heat shock response was transient for most of the tissues which synthesized the standard set of hsp. Hsp synthesis was detected up to 2 to 3 hours, but not at 10 hours of continuous 40°C treatment. The exception was suspension cultured cells, in which hsp synthesis showed only a slight reduction after 10 hours at 40°C. Tissue-specific differences in the heat-shock response suggest that there are differences in the way a given tissue is able to adapt to high temperature.

We have confirmed the previous suggestion that maize hsp do not accumulate in substantial quantities. Using two-dimensional gel analysis, hsp could be detected by autoradiography but not by sensitive silver staining techniques.

     

Structural proteins of simian virus 40. I. Histone characteristics of low-molecular-weight polypeptides.

The DNA-associated polypeptides of simian virus 40 (SV40), VP4 (mol wt 14,000), VP5 (mol wt 12,000), and VP6 (mol wt 11,000), have several properties characteristic of cell histones. After extraction from purified SV40 with dilute acids, these three polypeptides co-electrophoresed on low pH polyacrylamide gels with monkey-kidney cell histones F3, F2b, and F2a1. No virus polypeptide co-electrophoresed with histone F1. Polypeptides VP4, 5, and 6 lacked tryptophan, and only VP4 contained cysteine, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis of virus labeled in vivo with (3H)lysine and either (14C)tryptophan or (35S)cystine. All of the capsid polypeptides VP1, 2, and 3 contained tryptophan whereas only VP1 and 2 contained cysteine. In addition, VP4, 5, and 6 are rich in arginine and lysine when compared with virus labeled with a mixture of amino acids. Analysis of virus grown in cells labeled prior to infection showed that VP4, 5 and 6 were labeled fivefold greater than the major capsid polypeptide, VP1, which indicates that they were partially derived from preexisting cell histones. Based on these data and on previously determined molecular weight estimates, we conclude that VP4, 5, and 6 are histones F3, F2b, and F2a1, respectively, although the possibility that SV40 contains a small amount of F2a2 could not be excluded.     

Free messenger ribonucleoprotein complexes of chicken primary muscle cells following modification of protein synthesis by heat-shock treatment

When primary cultures of chicken myoblasts were subjected to incubation at a temperature higher than their normal growing temperature of 36-37 degrees C, the pattern of protein synthesis was altered. This condition of heat shock induced a vigorous production of a number of proteins collectively known as 'heat-shock proteins'. The synthesis of heat-shock proteins was achieved without a significant decrease in the production of a broad spectrum of proteins by muscle cells. The synthesis of three major heat-shock polypeptides with Mr values of 81 000, 65 000 and 25 000 was observed in both mononucleated dividing myoblast cells and terminally differentiated myotubes. Two-dimensional electrophoretic separation of the heat-induced polypeptides synthesized by myogenetic cultures further established that same set of polypeptides with Mr of 65 000 (pI 6.0 and 5.5), 81 000 (pI 6.2) and 25 000 (pI 5.6 and 5.3) were produced in myoblasts and myotubes. The effect of the changes in pattern of protein synthesis on the mRNA and protein moieties of non-polysomal cytoplasmic mRNA-protein complexes (free mRNP) was examined. Free mRNP complexes sedimenting at 20-35 S were isolated from the post-ribosomal supernatant of both normal and heat-shocked myotube cultures by centrifugation in a sucrose gradient. A 10-20S RNA fraction isolated from these complexes stimulated protein synthesis in a cell-free system. The RNA fraction obtained from heat-shocked cells appeared to direct the synthesis of all three major heat-shock proteins. In contrast, synthesis of these polypeptides was not detected when RNA from free mRNP complexes of normal cells was used for translation. The free mRNP complexes of both normal and heat-shocked cells showed a buoyant density of 1.195 g/cm3 in metrizamide gradients. A large number of polypeptides of Mr = 35 000-105 000 were present in the highly purified free mRNP complexes isolated from the metrizamide gradient. Similar sets of polypeptides were found in these complexes from both normal and heat-shocked myotube culture. However, the relative proportion of a 65 000-Mr polypeptide was dramatically increased in the free mRNP complexes of heat-shocked cells. Two-dimensional gel electrophoretic analysis revealed that this polypeptide and the 65 000-Mr heat-shock polypeptide exhibit similar electrophoretic migration properties. These observations suggest that, following heat-shock treatment of chicken myotube cultures, the changes in the pattern of protein synthesis is accompanied by alteration of the mRNA and protein composition of free mRNP complexes.     

Histone synthesis and replacement during spermatogenesis in the mouse.

Separation of labelled nuclei by sedimentation velocity at unit gravity (Staput method) was used to study the timing of histone synthesis and replacement by testis-specific basic nuclear protein (TSP) during spermatogenesis in the mouse. Animals were injected (intratesticularly) with 1.25 micronCi per testis 3H-arginine or 2.5 micronCi per testis 3H-lysine, testis nuclei were separated, and the acid extract of each nuclear fraction was analyzed by acrylamide gel electrophoresis. The distribution of labelled histones and TSP in separated nuclei was assessed 2 h after incorporation. Changes in the labelled histone and TSP content of nuclei during subsequent differentiation (1--34 days post-label) was followed in fractions of separated testis cell nuclei and in nuclei of cauda epididymal spermatozoa. Analysis of total histone and (TSP) content indicated quantitative changes during development. Nuclei from primary spermatocytes had relatively larger amounts of histones H1 and H4. Spermatid nuclei showed a relative reduction in histones H1 and H4, coincident with the appearance of TSP in these nuclei. These results suggested that synthesis and/or removal of certain histones must occur in late primary spermatocyte and early spermatid stages of spermatogenesis. Results of labelling experiments indicated several periods of histone synthesis during spermatogenesis: (1) closely associated with the last DNA synthesis(i.e., in early primary spermatocytes), (2) late in meiotic prophase (i.e., in pachytene primary spermatocytes) and (3) simultaneous with TSP synthesis (i.e., in late spermatids). Histone H1 was more heavily labelled toward the end of the primary spermatocyte period. Histone H4 was more heavily labelled in the early primary spermatocyte period, and again at the time of TSP synthesis in spermatids. Histones synthesized before the pachytene primary spermatocyte stage appeared to be replace, but histones synthesized later in spermatogenesis appeared to be at least partially retained in epididymal spermatozoa. These results suggested that repeated specific alterations in the protein complement of the nucleus are an integral part of spermatogenic differentiation in the mouse.     

Co-existence of two different types of soluble histone complexes in nuclei of Xenopus laevis oocytes

The nuclear pool of soluble histones in Xenopus laevis oocytes is organized into two major types of acidic histone complexes separable by sucrose density gradient centrifugation. One type of complex sediments at 5 S (Mr approximately 120,000), is isoelectric at pH 4.6, and contains histones H3 and/or H4 tightly bound to one polypeptide of a pair of very acidic polypeptides, designated N1 and N2 (Kleinschmidt, J. A., and Franke, W. W. (1982) Cell 29, 799-809). This complex can be selectively immunoprecipitated by guinea pig antibodies against purified protein N1/N2. In contrast, a larger complex of 7 S contains four histones and nucleoplasmin (the purified protein exists as a pentamer of a polypeptide of Mr approximately 30,000), is isoelectric over the pH range of 5-7, and can be immunoprecipitated by nucleoplasmin antibodies. Its relative molecular weight of 130,000-170,000, as determined by gel filtration, sucrose density gradient centrifugation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cross-linked complexes, excludes the association of a histone octamer with nucleoplasmin. In addition to histones H2A and H2B, two histones (designated H3 and H4) which are similar in their electrophoretic mobilities to histones H3 and H4 but have lower isoelectric pH values are enriched in immuno-precipitates obtained with nucleoplasmin antibodies. Cross-linking of complexes present in intact nuclei, using 1% formaldehyde at near-physiological ionic strength and pH, indicates the coexistence of these two soluble histone complexes in the living cell. In chromatin assembly experiments using SV 40 DNA, both histone fractions are able to transfer histones to DNA, resulting in an increase of DNA superhelicity and the formation of beaded nucleoprotein complexes of nucleosome-like morphology. The common principle governing both types of complexes, i.e. the association of one or two histone molecules with a karyophilic large acidic histone-binding protein is emphasized. We discuss the possible role of these complexes in storing histones utilized in chromatin assembly during early amphibian embryogenesis as well as the possible existence of similar complexes, albeit at lower concentrations, in somatic cells.     

Effect of day of the oestrous cycle, side of the reproductive tract and heat shock on in-vitro protein secretion by bovine endometrium

Endometrial explant cultures were prepared from 16 Brahman x Angus cows killed on Days 0, 2, 5 or 8 after oestrus. Cultures proceeded for 24 h at 39 degrees C (homeothermic) or 43 degrees C (heat shock) in a modified Eagle's minimal essential medium supplemented with 50 microCi L-[4,5(-3)H]leucine. Analysis by two-dimensional polyacrylamide gel electrophoresis of de-novo synthesized proteins secreted into the medium indicated that the major types of secreted polypeptides did not change over Days 0-8. Nevertheless, overall endometrial secretion of protein (incorporation of [3H]leucine into non-dialysable radioactivity in culture supernatants) was greatest at Day 0 and declined thereafter. Incorporation of [3H]leucine into TCA-precipitable material in tissue homogenates was also greatest at Day 0. For tissue cultured at 39 degrees C, several individual polypeptides were secreted at greater rates by endometrium from the horn of the uterus ipsilateral to the corpus luteum, with side differences tending to be greatest at Day 0 or Day 2. Overall, secretion of de-novo synthesized protein by endometrium was significantly elevated by heat shock at Day 0, but not affected thereafter. Nonetheless, heat shock reduced secretion of several individual proteins and exhibited interactions with day of the oestrous cycle and with side of the uterus. Secretion of 7 polypeptides was reduced by heat shock in tissue from the ipsilateral horn of the uterus but not in endometrium from the contralateral horn. We suggest that endometrial protein secretion changes quantitatively during the early oestrous cycle. In addition, there is a local influence of the ovary bearing the corpus luteum on endometrial function that may be disrupted by heat shock.     

Biosynthesis of specific histones during meiotic prophase of mouse spermatogenesis

A kinetics study has demonstrated histone synthesis occurring at two distinct phases during meiotic prophase of mouse spermatogenesis. These two periods have been delineated by quantifying the synthesis of DNA and basic nuclear proteins in spermatogenic cells at discrete intervals following the intratesticular injection of [3H] thymidine and [14C] arginine, respectively. One phase of histone synthesis occurs coincident with DNA synthesis in preleptotene spermatocytes. By contrast, a second phase of histone synthesis occurs during midprophase of meiosis, independent of semiconservative DNA synthesis. The [14C] arginine incorporated into the basic nuclear proteins of pachytene spermatocytes is conserved during spermiogenesis and then subsequently discarded within the residual bodies, which are formed during late spermiogenesis. Fluorographic analyses of isotopically labeled basic nuclear proteins in pachytene spermatocytes has shown that only the somatic complement of histones are synthesized during the preleptotene period, whereas the second phase involves the synthesis of proteins H1t, H2S, and "A". In addition, several nonhistone basic nuclear proteins are synthesized concomitant with the germ cell-specific histones. Thus, the data clearly demonstrate that pachytene spermatocytes actively synthesize a number of novel chromatin-associated polypeptides.     

Resolution of basic cellular proteins including histone variants by two-dimensional gel electrophoresis: Evaluation of lysine to arginine ratios and phosphorylation

A procedure is described which resolves histones and other very basic cellular polypeptides from solubilized whole cells by two-dimensional gel electrophoresis. The entry of histones into the gel was apparently quantitative when salt and protamine were added to solubilization buffers containing urea and detergents. Histones and basic polypeptides in the histone region of the gels were identified and characterized by comparison with purified histones and by determining lysine to arginine ratios of individual spots. Phosphorylated derivatives of the nucleosome histones were clearly resolved from stained spots in the charge dimension. Some phosphorylated H1's comgrated with the stained spots and some were retarded in the charge dimension. Acetylated nucleosome histones were nearly coincident with stained spots. The usefulness of this technique for evaluating changes in post-translational modification of histones was illustrated by showing that one H1-like protein increased in ³²P content when C-6 cells were treated with a β-adrenergic agonist.     

A comparison of the chilling-stress response in two differentially tolerant cultivars of tomato (Lycopersicon esculentum).

The chilling responses of two differentially cold tolerant cultivars of tomato were monitored through in vivo labelling of polypeptides with [35S]methionine, both during a gradual temperature decrease (2 degrees C/day) and also during a rapid cold shock (4 degrees C). The polypeptides were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and revealed by fluorography. Both cultivars showed changes in the polypeptide profiles resulting from either chilling treatment. During the gradual temperature decrease, there were few differences exhibited between the two cultivars. However, during cold shock both cultivars showed the altered synthesis of several unique polypeptides. Both cultivars showed the appearance of a 35-kDa polypeptide during the gradual temperature decrease and also during the cold shock. The appearance of three high relative mass polypeptides was found in both cultivars only during the gradual temperature decrease. Treatments with cycloheximide and chloramphenicol suggested that cold-shock polypeptides are both nuclear and organelle encoded. The cold-shock response in roots was different from the response in leaves and between cultivars. A comparison of the two cultivars showed a number of differences in polypeptide synthesis which may be related to increased cold tolerance.     

Heat-Shock Response in Heat-Tolerant and Nontolerant Variants of Agrostis palustris Huds

The heat-shock response in heat-tolerant variants (SB) and non-tolerant variants (NSB) of creeping bentgrass (Agrostis palustris Huds.) was investigated. Both variants were derived from callus initiated from a single seed of the cultivar Penncross. SB and NSB synthesized heat-shock proteins (HSPs) of 97, 83, 70, 40, 25, and 18 kD. There were no major differences between SB and NSB in the time or temperature required to induce the heat-shock response. When the HSPs synthesized by SB and NSB were analyzed by two-dimensional gel electrophoresis, it was apparent that SB synthesized two to three additional members of the HSP27 family, which were smaller (25 kD) and more basic than those synthesized by NSB. Analysis of F1 progeny of NSB x SB indicated that 7 of the 20 progeny did not synthesize the additional HSP25 polypeptides. These progeny were significantly less heat tolerant than progeny that did synthesize the additional HSP25 polypeptides. The X2 test of independence (X2 = 22.45, P < 0.001) indicated that heat tolerance and the presence of the additional HSP25 polypeptides are linked traits.     

Lack of heat-shock response in preovulatory mouse oocytes

The response to heat (hs response) of preovulatory mouse oocytes was compared with that of mouse granulosa cells and characterized in regard to in vitro resumption of meiosis, amino acid incorporation into total protein, and qualitative analysis of protein synthesized before and after the shock. Granulosa cells displayed a hs response typical of other mammalian systems. When incubated at 43 degrees C for 20-40 min, these cells maintained a normal level of amino acid incorporation into total protein, responded to stress by new synthesis of 33- and 68-kDa heat-shock proteins (hsps), and enhanced synthesis of 70-kDa heat-shock cognate protein (hsc70) and of 89- and 110-kDa hsps. In contrast to granulosa cells, preovulatory mouse oocytes were very sensitive to hyperthermia. Incubation at 43 degrees C for 20-40 min strongly inhibited oocyte resumption of meiosis and protein synthesis and did not induce a new or enhanced synthesis of hsps. Unstressed preovulatory mouse oocytes constitutively synthesized 70- and 89-kDa polypeptides resembling hsc70 and hsp89 of granulosa cells.