Isolation of histone-like proteins from mitochondria of bovine heart

Two methods for isolating and purifying histone-like proteins from mitochondria of bovine heart are described. In the first, a sonicated extract of the mitochondria was fractionated in three chromatography steps, including affinity chromatography on DNA-cellulose, to purify a protein that resembles very closely the histone-like protein (HM) of yeast mitochondria. In the second method, an acid extract of the heart mitochondria was the starting material; two other histone-like proteins were separated. Thus, as in mitochondria of Xenopus laevis, several histone-like proteins are present in mitochondria of bovine heart.     

Mitochondrial histone-like DNA-binding proteins are essential for normal cell growth and mitochondrial function in Crithidia fasciculata

The Crithidia fasciculata KAP2 and KAP3 proteins are closely related kinetoplast-specific histone-like DNA-binding proteins. The KAP2 and KAP3 genes are 46% identical and are arranged in tandem on the chromosomal DNA. Disruption of both alleles of either gene alone shows no detectable phenotype. However, replacement of both copies of the sequence encoding the entire KAP2 and KAP3 locus increases maxicircle mRNA levels two- to fourfold. These double-knockout cells are viable but grow extremely slowly, have reduced respiration and very abnormal cell morphologies, and accumulate numerous large vacuoles. The extreme phenotype of these mutant cells suggests an important role for the KAP2 and KAP3 proteins in mitochondrial metabolism and cell growth.     

Evolutionary origin of the protozoan parasites histone-like proteins (HU)

The histone-like proteins (HU) belong to a family of DNA architectural proteins that stabilize nucleoprotein complexes. We found a putative HU protein (TgGlmHMM_3045) in Toxoplasma gondii genome that was homologous to the bacterial HU protein. This putative sequence was located in the scaffold TGG_995361 of the chromosome 10. The sequence included the prokaryotic bacterial histone-like domain, KFGSLGlRRRGERVARNPRT (ID number PS00045). HU protein sequences were also found in Plasmodium falciparum, Neospora caninum, Theileria parva and Theileria annulata. We found that the homology of the putative HU protein in Apicomplexa was greater with bacterial histone-like proteins than with eukaryotic histone proteins. The phylogenetic tree indicated that the putative HU protein genes were acquired in Apicomplexa by means of a secondary endosymbiotic event from red algae and later they were transferred from the apicoplast organelle to the nuclear genome.     

Archaebacterial histone-like proteins. Purification and characterization of helix stabilizing DNA binding proteins from the acidothermophile Sulfolobus acidocaldarius

Four DNA binding histone-like proteins have been purified from the nucleoid of the acidothermophilic archaebacterium Sulfolobus acidocaldarius to homogeneity employing DNA-cellulose chromatography and carboxymethylcellulose chromatography. The molecular weights of these proteins are in the range 8,000-12,500. Immunoblotting results suggest that a few antigenic determinants are common among these proteins which could not be detected by immunodiffusion. Spectroscopic properties of the proteins have been studied. The amino acid compositions of these proteins show both similarities and differences with histones and prokaryotic histone-like proteins. All of the four proteins bind native and denatured DNAs and single stranded RNA with differing affinities. Three of the proteins, denoted by HSNP (helix stabilizing nucleoid protein)-A, HSNP-C, and HSNP-C', show physiologically significant, strong, and synergistic effects in stabilizing duplex DNA against thermal denaturation with Tm increases in the range of 15-30 +/- degrees C.     

Regulation of gene expression by histone-like proteins in bacteria

Histone-like proteins in bacteria contribute to the control of gene expression, as well as participating in other DNA transactions such as recombination and DNA replication. They have also been described, somewhat vaguely, as contributors to the organization of the bacterial nucleoid. Our view of how these proteins act in the cell is becoming clearer, particularly in the cases of Fis, H-NS and HU, three of the most intensively studied members of the group. Especially helpful have been studies of the contributions of these proteins to the regulation of specific genes such as the gal operon, and genes coding for stable RNA species, topoisomerases, and the histone-like proteins themselves. Recent advances have also been assisted by insights into the effects the histone-like proteins exert on DNA structure not only at specific promoters but throughout the genome.     

A Systematic In Vitro Study of Nucleoprotein Complexes Formed by Bacterial Nucleoid-Associated Proteins Revealing Novel Types of DNA Organization

Bacterial nucleoid is a dynamic entity that changes its three-dimensional shape and compaction depending on cellular physiology. While these changes are tightly associated with compositional alterations of abundant nucleoid-associated proteins implicated in reshaping the nucleoid, their cooperation in regular long-range DNA organization is poorly understood. In this study, we reconstitute a novel nucleoprotein structure in vitro, which is stabilized by cooperative effects of major bacterial DNA architectural proteins. While, individually, these proteins stabilize alternative DNA architectures consistent with either plectonemic or toroidal coiling of DNA, the combination of histone-like protein, histone-like nucleoid structuring protein, and integration host factor produces a conspicuous semiperiodic structure. By employing a bottom-up in vitro approach, we thus characterize a minimum set of bacterial proteins cooperating in organizing a regular DNA structure. Visualized structures suggest a mechanism for nucleation of topological transitions underlying the reshaping of DNA by bacterial nucleoid-associated proteins.     

Purification and properties of the antizymes of Escherichia coli to ornithine decarboxylase

The purification of the antizymes to ornithine decarboxylase of Escherichia coli to homogeneity is detailed. An acidic component, pI 3.8, and two basic histone-like proteins, pI above 9.5, are described. The two latter proteins constitute approximately 90% of the total antizyme activity.     

Comparative aspects of basic chromatin proteins in dinoflagellates

Previous work on histone-like proteins in dinoflagellates is summarized, together with some new data to give an overview of basic proteins in these algae. The first two dinoflagellates studied were both found to contain one major acid-soluble protein that migrated to the same position in acidic-urea gels. When several other genera were studied however, it became apparent that the histone-like proteins from different dinoflagellates were similar but not identical. In view of the great diversity of living dinoflagellates it is speculated that further differences in dinoflagellate basic chromatin proteins will be revealed. Electrophoretic data from the eukaryotic (endosymbiont) nucleus of Peridinium balticum showed the presence of five major components. It is speculated that two of these proteins represent an H1-like doublet and two others correspond to the highly conserved histones H3 and H4. The fifth component is a new histone that may substitute for H2A and H2B in the nucleosome. Because histones and nucleosomes are present in all higher organisms but completely lacking in procaryotes, studies on basic proteins in dinoflagellates will provides insights into the evolution of histones and eucaryotic chromatin organization.     

Characterization of the unusual basic proteins of cricket spermatid nuclei on the basis of their molecular weights and amino acid compositions

Typical somatic cell type histones are lost from the nucleus during late spermiogenesis in the house cricket; they are replaced by unusual basic proteins specific to the spermatid. We wish to characterize these proteins because they appear to determine the unusual chromatin structures of the spermatid. Molecular weights of the unusual basic proteins were estimated by chromatographing them on Bio-Gel A 0.5 M agarose columns eluted with 6 M guanidine hydrochloride. Two proteins named TH1 and TH2 have molecular weights in the range spanned by the somatic histones. The molecular weight of TH1 is 17 500 and that of TH2 is 15 500. Three additional spermatid proteins were also analyzed by molecular weight determination. They are called here protamines A, B and C, and they have molecular weights in the range typical of protamines. That of A is 6200, of B is 5500 and of C is 3800. They span the range from the large protamines typical of mammalian sperm to the small protamines of salmonid fish. The molecular weights of the TH proteins were also examined by electrophoresis on SDS-polyacrylamide gels. Amino acid compositions determined for TH1 and TH2 show that both are basic proteins rich in arginine relative to lysine. Their compositions are histone-like, but they appear to be distinct histone types rather than variant forms of the somatic histones.     

Structure-function relationship and regulation of two Bacillus subtilis DNA-binding proteins, HBsu and AbrB

Microorganisms use a number of small basic proteins for organization and compaction of their DNA. By their interaction with the genome, these proteins do have a profound effect on gene expression, growth behavior, and viability. It has to be distinguished between indirect effects as a consequence of the state of chromosome condensation and relaxation that influence the rate of RNA polymerase action as represented by the histone-like proteins, and direct effects by specific binding of proteins to defined DNA segments predominantly located around promoter sequences. This latter class is represented by the transition-state regulators that are involved in integrating various global stimuli and orchestrating expression of the genes under their regulation for a better adaptation to changes in growth rate. In this article we will focus on two different but abundant DNA binding proteins of the gram-positive model organism Bacillus subtilis, the histone-like HBsu as a member of the unspecific and the transition state regulator AbrB as a member of specific classes of DNA binding proteins.     

Novel histone-like DNA-binding proteins in the nucleoid from the acidothermophillic archaebacterium Sulfolobus acidocaldarius that protect DNA against thermal denaturation

DNA of acidothermophilic archaebacterium Sulfolobus acidocaldarius has a base composition of about 40 mol% G + C content. A low intracellular salt concentration has been inferred for this organism. These features and the high optimal temperature of growth (75°C) would have a destabilising effect on the helical structure of the intracellular DNA. Hence, the nucleoid of this organism has been isolated in order to analyse its proteins composition and to identify any protein factors responsible for stabilisation of the organism's DNA at its growth temperature. The acid-soluble fraction of the nucleoid contains four low-molecular-weight basic proteins. The four proteins have been purified to homogeneity and antibodies to these proteins have been raised in rabbits. Immunodiffusion results suggest that the proteins are antigenically distinct. Three proteins (A, C and C′) stabilise different double-stranded DNA during thermal denaturation and increase T_m of DNA by about 25 C°. These proteins are referred to as helix-stabilising nucleoid proteins (HSNP). Protein B (referred to a DNA-binding nucleoid protein, DBNP-B) does not show helix-stabilising effect. None of the four proteins stabilises double-stranded RNA. The four proteins bind to native and denatured DNA to different extents as measured by DNA-cellulose chromatography and [³H]DNA binding by filtration. We suggest, based on the DNA binding, histone-like and helix-stabilising properties, that the intracellular function of these proteins is to prevent strand separation of DNA at the optimal temperature of growth (75°C).     

Histone-like proteins of bacteria (review)

Four major families of bacterial histone-like proteins (HU, IHF, H-NS, FIS), united on the basis of structural similarity and performing specific structural and regulatory functions in the cell, are discussed. Histone-like proteins perform topological modification of the chromosome (twisting, bending, and folding) and directly regulate the functioning of promoters of individual operons. Histone-like proteins are critical for the regulation of cell metabolism, are involved in the response to environmental changes, and play a key role in the transition to and maintenance of the resting cells of bacteria.     

Alveolata histone-like proteins have different evolutionary origins

Prokaryotic histone-like proteins (Hlps) are abundant proteins found in bacterial and plastid nucleoids. Hlps are also found in the eukaryotic dinoflagellates and the apicomplexans, two major lineages of the Alveolata. It may be expected that Hlps of both groups were derived from the same ancestral Alveolates. However, our phylogenetic analyses suggest different origins for the dinoflagellate and the apicomplexan Hlps. The apicomplexan Hlps are affiliated with the cyanobacteria and probably originated from Hlps of the plastid genome. The dinoflagellate Hlps and the proteobacterial long Hlps form a clade that branch off from the node with the proteobacterial short Hlps.     

Mitochondrial nucleoid staining with ammoniacal silver

Under controlled conditions, ammoniacal silver (A-S) reaction appears in the nucleoid in the mitochondria of Physarum polycephalum as well as the dense chromatin in the nuclei. The results and the effect of selective extraction of basic proteins on the A-S reaction of the nucleoid and the nucleus suggest that the mitochondrial nucleoid may contain basic protein, probably histone-like protein.     

CHARACTERIZATION OF GYMNODINIUM MIKIMOTOI (DINOPHYCEAE) NUCLEI AND IDENTIFICATION OF THE MAJOR HISTONE-LIKE PROTEIN, HGm

A simple and rapid method for isolation of nuclei from Gymnodinium mikimotoi Miyake et Kominami ex Oda is described along with chemical characterization of the nuclei. The isolated nuclei were completely free of whole cells, 99.96% free of cytoplasmic contamination, and were collected with a yield of 40% from harvested whole cells. Each nucleus contained 47 pg of DNA and the ratio of DNA to acid-soluble proteins to acid-insoluble proteins was 1:0.25:1.21, respectively. SDS electrophoresis of acid-extracted proteins showed one histone-like protein, which we termed HGm, with an apparent molecular mass of 12 kDa. V8 protease digestion analysis of HGm, the histone-like protein from Crypthecodinium cohnii (HCc), and two histone-like proteins from Gymnodinium dorsum , showed that the HGm digestion pattern was more similar to that of HCc than to that of either of the G. dorsum histone-like proteins.     

HMG proteins (1 + 2) form beaded structures when complexed with closed circular DNA.

Structures bearing a resemblance to nucleosomes can be assembled by incubating calf thymus High Mobility Group proteins (1 + 2) with closed circular DNA. These HMG proteins are capable of forming beads and inducing superhelicity when bound to DNA. However, they do not protect from nuclease digestion the discrete DNA fragments characteristic of nucleosomes. The relationship between HMGs (1 + 2) and the "primitive" histone-like DNA-packaging proteins from prokaryotes and mitochondria is discussed.     

The physiological role of eubacterial histone-like proteins

This minireview reflects the change in views on the role of histone-like proteins which has occurred in the 1980 s. Initially these proteins were regarded as analogous to eukaryotic histones though distinguished from the latter by much lower strength of binding to DNA. This was attributed to the greater dynamics of the structure of bacterial chromatin. The accumulation in recent years of results testifying to the absence of protein HU in central region of the nucleoid and the participation of this protein in almost all processes involving the recognition of specific DNA sequences by regulatory proteins forces the rejection of the concept of "histone-likeness" and favors the concept of a general, non-specific enhancer of the recognition of sequences acting by causing changes in the secondary structure of a given DNA region.