Molecular cloning and immunolocalization of two variants of the major basic nuclear protein (HCc) from the histone-less eukaryote Crypthecodinium cohnii (Pyrrhophyta)

Two clones that encode variants (HCc1 and HCc2) of the major basic nuclear protein of the dinoflagellate Crypthecodinium cohnii, were identified by immunoscreening of a cDNA expression library. The first clone carries a full-length cDNA with an open reading frame (HCc1) encoding 113 amino acids. The cDNA from the second clone lacks some of the 5 end, and the coding sequence is only 102 residues. The two proteins display 77% sequence similarity and their NH₂-ends are homologous to the NH₂-peptide of the HCc protein determined by P. Rizzo. The amino acid composition, which confirms the basic nature of lysine-rich HCc proteins, differs markedly from other known DNA-binding proteins such as histones, HMGs or prokaryotic histone-like proteins. No convincing homology was found with other proteins. HCc antigens were localized on C. cohnii by immunofluorescence, and by electron microscopy (EM) with immunogold labelling. HCc proteins are mainly detected at the periphery of the permanently condensed chromosomes, where active chromatin is located, as well as in the nucleolar organizing region (NOR). This suggests that these basic, non-histone proteins, with a moderate affinity for DNA, are involved at some level in the regulation of gene expression.by M. Trendelenburg     

Histone-like proteins of the dinoflagellate Crypthecodinium cohnii have homologies to bacterial DNA-binding proteins

The dinoflagellates have very large genomes encoded in permanently condensed and histoneless chromosomes. Sequence alignment identified significant similarity between the dinoflagellate chromosomal histone-like proteins of Crypthecodinium cohnii (HCCs) and the bacterial DNA-binding and the eukaryotic histone H1 proteins. Phylogenetic analysis also supports the origin of the HCCs from histone-like proteins of bacteria.     

Immunocytochemical localization of the DNA-binding protein HCc during the cell cycle of the histone-less dinoflagellate protoctista Crypthecodinium cohnii B

The major basic nuclear protein HCc (previously named Histone-like) of the dinoflagellate Crypthecodinium cohnii B was immunolocalized in light and electron microscopy using an affinity-purified polyclonal antibody. Complementary conventional and cryo-techniques were used to study the distribution of the DNA-binding protein in interphase cells and to follow its behaviour throughout the mitotic cycle. In non-dividing cells, the HCc protein was found to be located on extra-chromosomal loops and chromosomal nucleofilaments dispersed in the nucleoplasm. In mitotic cells, from prophase to early telophase, it was homogeneously distributed in the (whole) dividing chromosomes. HCc protein was also detected in two compartments of all the permanently observable nucleoli: the nucleolar organizing region and the fibrillo-granular region. In this paper we discuss the hypothetical roles, structural and/or functional, of this DNA-binding protein, which is specific to dinoflagellates, the only eukaryotes whose chromatin is devoid of histones and nucleosomes.     

Some properties of the histone-like protein from Crypthecodinium cohnii (HCc)

The histone-like protein from Crypthecodinium cohnii (HCc) was examined in regard to its amino acid composition and the peptide pattern resulting from protease digestion. A revised amino acid composition indicated a higher lysine and arginine content and a lower glycine content than that determined previously. Comparative peptide mapping of HCc with HTa, a histone-like protein from Thermoplasma acidophilum, and with a histone-like protein from the dinoflagellate Gyrodinium dorsum showed significant differences in the peptide patterns produced.     

Identification of the nuclear matrix and chromosome scaffold in dinoflagellate Crypthecodinium cohnii

Dinolflagellate is one of the primitive eukaryotes,whose nucleus may represent one of the transition stages from prokaryotic nucleoid to typical eukaryotic nucleus,Using selective extraction together with embeddment-free section and whole mount electron microscopy,a delicate nuclear matrix filament network was shown,for the first time,in dinoflagellate Crypthecodinium cohnii nucleus,Chromosome residues are connected with nuclear matrix filaments to form a complete network spreading over the nucleus,Moreover,we demonstrated that the dinoflagellate chromosome retains a protein scafflod after the depletion of DNA and soluble proteins.This scaffold preserves the characterstic morphology of the chromosome.Two dimensional electrophoreses indicated that the nuclear matrix and chromosome scaffold are mainly composed of acidic proteins.Our results demonstrated that a framework similar th the nuclear matrix and chromosome scaffold in mammalian cells appears in this primitive eukaryote,suggesting that these structures may have been originated from the early stages of eukaryote evolution.     

寇氏隐甲藻（Crypthecodinium cohnii）细胞核骨架与中间纤维...

Nuclear matrix (NM) and intermediate filament (IF) scaffold in primitive eukaryote Crypthecodinium cohnii were shown using selective extraction together with embedment-free electron microscopy, whole mount cell preparation and immunoblot techniques. There exists a delicate NM-IF network spreading over cytoplasm and nucleus in dinoflagellate cells, however, nuclear lamina is undeveloped. The diameter of NM fiber is about 3-5 nm and IF is 10 nm. Chromosomes are connected with NM filament network. Immunoblot analysis showed that dinoflagellate contained keratin-like polypeptides (63 kD and 67 kD) while mammalian lamin antibodies did not crossreact with dinoflagellate total protein. Our experiment results demonstrated that a framework similar to NM-IF scaffold in mammalian cell appeared in primitive eukaryote. We propose that: (1) NM-IF scaffold is not restrict to vertebrate cell, and it may be originated from early stages of eukaryote evolution; (2) Keratin is probably very conservative; (3) Compared with IF, lamina might appear late in evolution, and some of primitive characteristics of dinoflagellate nucleus may be related to the lack of lamina.     

Distribution of Members of the Crypthecodinium cohnii (Dinophyceae) Species Complex1

Crypthecodinium cohnii, a small marine heterotrophic homothallic dinoflagellate, has diversified into a complex of morphologically very similar breeding groups (biological species or sibling species), some of which have become widely dispersed. Membership of two clones in the same sibling species is shown by their sexual compatibility as determined by genetic complementation in zygotes formed from motility mutants derived from the two stocks. Membership in different sibling species may be inferrec when motility mutants of one strain do not complement those of another. Fifty-six clones representing seaweed enrichments from *** geographic sites have been found to belong to 28 sibling species; 35 clones are members of seven wide-ranging biological species, and 21 are single representatives of 21 other breeding groups within the ranges of the others. Of 174 clonal isolates in our possession, 168 conform in size and shape to C. cohnii. Six others which have smaller cells and only one-fifth the standard DNA and chromosome number belong, we believe, to another species. The C. cohnii complex provides a unique opportunity for the study of evolutionary divergence and geographical dispersion of a dinoflagellate.     

Characterization of the DNA from the dinoflagellate Crypthecodinium cohnii and implications for nuclear organization.

Although dinoflagellates are eucaryotes, they possess many bacterial nuclear traits. For this reason they are thought by some to be evolutionary intermediates. Dinoflagellates also possess some unusual nuclear traits not seen in either bacteria or higher eucaryotes, such as a very large number of identical appearing, permanently condensed chromosomes suggesting polyteny or polyploidy. We have studied the DNA of the dinoflagellate Crypthecodinium cohnii with respect to DNA per cell, chromosome counts, and renaturation kinetics. The renaturation kinetic results tend to refute extreme polyteny and polyploidy as the mode of nuclear organization. This organism contains 55-60% repeated, interspersed DNA typical of higher eucaryotes. These results, along with the fact that dinoflagellate chromatin contains practically no basic protein, indicate that dinoflagellates may be organisms with a combination of both bacterial and eucaryotic traits.     

Electrophoretic Characterization of Members of the Crypthecodinium cohnii (Dinophyceae) Species Complex1

Eighty-seven axenic clones of the colorless inshore dinoflagellate Crypthecodinium cohnii were found by mating experiments to fall into 52 sibling species, seven wide ranging (two possibly global)—called major sibling species—and 45 found only once—minor sibling species. Electrophoretic analysis of three soluble enzymes from these strains revealed the following: 1) Despite some polymorphism most members of major sibling species closely resemble one another electrophoretically. 2) Major sibling species and most minor ones are electrophoretically distinct. 3) Sharing of electromorphs is sufficiently extensive, however, that no major sibling species is totally unrelated to all others. 4) Some minor sibling species are electrophoretically indistinguishable from a member of a major sibling species or from one another, suggesting recent origin by sexual isolation in situ. 5) Other minor sibling species differ from majors by one, two, or all three of the enzymes studied. A “model” of sexual isolation and diversification is offered.     

Presence of poly (ADP-ribose) polymerase and poly (ADP-ribose) glycohydrolase in the dinoflagellate Crypthecodinium cohnii

Poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase have been detected in chromatin extracts from the dinoflagellate Crypthecodinium cohnii. Poly(ADP-ribose) glycohydrolase was detected by the liberation of ADP-ribose from poly(ADP-ribose). Poly(ADP-ribose) polymerase was proved by (a) demonstration of phosphoribosyl-AMP in the phosphodiesterase digest of the reaction product, (b) demonstration of ADP-ribose oligomers by fractionation of the reaction product on DEAE-Sephadex. The (ADP-ribose)-protein transfer is dependent on DNA; it is inhibited by nicotinamide, thymidine, theophylline and benzamide. The protein-(ADP-ribose bond is susceptible to 0.1 M NaOH (70%) and 0.4 M NH2OH (33%). Dinoflagellates, nucleated protists, are unique in that their chromatin lacks histones and shows a conformation like bacterial chromatin [Loeblich, A. R., III (1976) J. Protozool. 23, 13--28]; poly(ADP-ribose) polymerase, however, has been found only in eucaryotes. Thus our results suggest that histones were not relevant to the establishment of poly(ADP-ribose) during evolution.     

N-methyl-N'-nitro-N-nitrosoguanidine and UV induced mutants of the dinoflagellate Crypthecodinium cohnii

Mutant strains were chemically induced by treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and UV irradiation. UV and NTG mutation rates were obtained that were both consistent with the organism being haploid. Three types of mutants were produced: (a) strains deficient in both beta- and gamma-carotene, the only carotenoids found in the wild type; phenotypes include albinos (translucent, dull white, "snow white") and cream-colored on agar as compared to the yellow-orange color of wild type colonies; (b) strains requiring adenine, guanine or cytosine in addition to the minimal medium for growth; (c) mutants that grow at a rate less than 40% of the wild type in minimal medium.     

Composition and DNA-binding properties of the nuclear matrix proteins from mammalian cell nuclei

A rapidly sedimenting DNA-protein complex was isolated from nuclear lysates in 2 M NaCl and characterized with regard to its polypeptide composition and the DNA-binding properties of the purified proteins. The complex consists of the nuclear matrix with attached DNA. Electrophoresis in SDS-polyacrylamide gels revealed two major and five minor polypeptide bands, mainly in the 60 to 75 kDa molecular weight region. The DNA-matrix complex dissociated into free DNA and proteins in the presence of 2 M NaCl and 5 M urea. The proteins could be purified by chromatography on hydroxyapatite and showed a strong tendency to reassociate at 0.15 M NaCl concentration in the absence of urea. DNA was bound to the reassociated proteins at 0.15 M NaCl concentration. Part of the DNA-protein complex was stable at 1 M NaCl concentration. The binding appeared to be random with regard to the DNA sequence.     

The in vitro DNA-binding properties of purified nuclear lamin proteins and vimentin

The ability of purified nuclear lamin A, lamin B, lamin C, and vimentin from Ehrlich ascites tumor cells to bind nucleic acids was investigated in vitro via a quantitative filter binding assay. At low ionic strength, vimentin bound more nucleic acid than the nuclear lamins and showed a preference for G-containing nucleic acids. Nuclear lamins A and C were quite similar in their binding properties and bound G- and C-containing nucleic acids preferentially. The binding of poly(dT) by the lamins A and C was reduced in competition experiments by both poly(dG) and poly(dC), but not by poly(dA). Lamin B bound only oligo and poly(dG); no other nucleic acids tested were bound or could compete with the binding of oligo(dG). Vimentin, lamin A, and lamin C specifically bound a synthetic oligonucleotide human (vertebrate) telomere model. The Ka for vimentin (2.7 X 10(7) M-1) was approximately 10-fold higher than those for lamin A (2.8 X 10(6) M-1) and lamin C (2.9 X 10(6) M-1). Lamin B did not bind detectable amounts of the telomere model. Washing of lamin A- and lamin C-nucleic acid complexes, formed at low ionic strength, with solutions containing 150 mM KCl resulted in the elution of 30% of bound poly(dG)12-18 and 70% of bound synthetic oligonucleotide telomere model. These results, using purified individual proteins, are in good agreement with data from competition experiments with vimentin but are at odds with data obtained previously using a crude preparation of nuclear matrix proteins containing all three nuclear lamin proteins (Comings, D. E., and Wallack, A. S. (1978) J. Cell Sci. 34, 233-246). The nuclear lamins A and C and vimentin possess nucleic acid-binding properties that might permit their binding to specific base sequences and/or unique DNA structure, such as that observed for the binding of the telomere model. The significance of the higher affinity binding of nucleic acids by the cytoplasmic protein vimentin (compared with the nuclear lamins) remains to be elucidated.     

N-methyl-N′-nitro-N-nitrosoguanidine and UV induced mutants of the dinoflagellate Crypthecodinium cohnii*

SYNOPSIS. Mutant strains were chemically induced by treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and UV irradiation. UV and NTG mutation rates were obtained that were both consistent with the organism being haploid. Three types of mutants were produced: (a) strains deficient in both β- and γ-carotene, the only carotenoids found in the wild type; phenotypes include albinos (translucent, dull white, “snow white”) and cream-colored on agar as compared to the yellow-orange color of wild type colonies; (b) strains requiring adenine, guanine or cytosine in addition to the minimal medium for growth; (c) mutants that grow at a rate less than 40% of the wild type in minimal medium.     

The sensitivity of the dinoflagellate Crypthecodinium cohnii to transient hydrodynamic forces and cell-bubble interactions

The increased interest in the benefits of omega-3 fatty acids for human health has resulted in the commercial development of the dinoflagellate Crypthecodinium cohnii for production of docosahexaenoic acid (DHA). The growing market demand for DHA requires highly efficient, very large scale cultures of DHA. While the effects of hydrodynamic forces on dinoflagellates have been investigated for several decades, the majority of the work focused on the negative effects of oceanic turbulence on the population growth of environmentally important dinoflagellates. In contrast, significantly less is known on the effect of hydrodynamic forces encountered by algae in bioprocesses. Unlike other studies conducted on algae, this study employed a microfluidic, flow contraction device to evaluate the effect of transient hydrodynamic forces on C. cohnii cells. It was found that C. cohnii cells can sustain the energy dissipation rate of 5.8 x 10(7) W/m3 without lysis. However, an obvious sublethal effect, the loss of flagella, was observed at a lower level of 1.6 x 10(7) W/m3. Finally the cell-bubble interaction and the effect of bubble rupture were also explored to simulate the conditions of sparged bioreactors.     

Two MAR DNA-binding proteins of the pea nuclear matrix identify a new class of DNA-binding proteins

Four MAR-binding proteins of 60, 65, 70 and 72 kDa have been detected by South-Western blotting and isolated from pea nuclear matrices. Two cDNAs encoding the 60 and 65 kDa proteins (MARBP-1 and MARBP-2) were isolated from a pea leaf cDNA library by screening with a PCR product obtained using degenerate primers based on an amino acid sequence from the 60 kDa protein. The proteins of 560 and 550 amino acids are 86% identical and contain several KKD/E repeats near the C-terminus. Escherichia coli-expressed MARBP-1 specifically binds A/T-rich MAR DNA. The interaction of MARBP-1/MARBP-2 with MAR DNA involves novel DNA-binding motifs. The MARBP-1 and MARBP-2 genes are expressed in a range of pea tissues and are encoded by genes at different loci. MARBP-1 and MARBP-2 are homologous to yeast nucleolar proteins Nop56p and Nop58p, which are involved in ribosome biogenesis, and to similar highly conserved proteins in other eukaryotes and in archaebacteria. MARBP-1 and MARBP-2 may have multifunctional roles in chromatin organisation and ribosome biogenesis.     

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.     

Ribosomal RNA Sequencing of Members of the Crypthecodinium cohnii (Dinophyceae) Species Complex; Comparison with Soluble Enzyme Studies

ABSTRACT. Sixty-five members of the Ctypthecodinium cohnii species complex were analyzed for sequence differences within the D2 region of the 23S ribosomal RNA molecule. On the basis of 46 sequence differences the strains fell into 19 distinct ribosets (strains of identical sequence), some with many members. Members of four of the seven major sibling species (widespread breeding groups) were each found within single ribosets. Members of three other major sibling species were each, however, divided into two ribosets by a single sequence difference correlated with geographic separation and with previously reported electrophoretic polymorphisms of soluble enzymes within the sibling species. In addition to members of major sibling species, some ribosets include many minor sibling species (each represented by only one strain). Of 38 minor sibling species, 22 shared sequence with a major sibling species. Of these 22, 14 were identical in soluble enzymes to their related major sibling species or differed by only one of three enzymes. Other minor sibling species appear to have diverged extensively from any others in both rRNA sequence and electrophoretic profile. As a group, major sibling species differ markedly in the number of minor sibling species associated with them, suggesting differences in frequency of sexually isolating events in their past histories. These findings are discussed in the context of the previously proposed model of sympatric speciation.