DNA (cytosine-5) methyltransferase turnover and cellular localization in developing Paracentrotus lividus sea urchin embryo.

The turnover and localization of the enzyme DNA (cytosine-5) methyltransferase (Dnmt1) were studied during Paracentrotus lividus sea urchin embryo development using antibody preparations against the NH(2) and COOH-terminal regions of the molecule. The antibodies reveal, by Western blots and whole-mount analyses, that the enzyme is differently required during embryonic development. The changeover point is at blastula stage, where a proteolytic mechanism hydrolyses the enzyme present in all embryonic cells by removing a peptide of about 45 kDa from the amino terminal region of the 190 kDa enzyme initially synthesized on maternal transcripts. The resulting 145 kDa enzyme shows modified catalytic properties, different antibody reactivity and is rapidly destroyed in the few hours before gastrulation. At more advanced stages of development the enzyme is newly synthesized but only in particular cell types, among which neurons. The data show that Dnmt1 is removed from embryonic cells before gastrulation to be synthesized again at different levels in different cell types, indicating that the concentration of Dnmt1 is critical for the various differentiated cells of the developing sea urchin embryo.

Characterization of a new variant DNA (cytosine-5)-methyltransferase unable to methylate double stranded DNA isolated from the marine annelid worm Chaetopterus variopedatus.

The enzyme S-adenosylmethionine-DNA (cytosine-5)-methyltransferase has been identified, first time for invertebrates, in embryos of the marine polychaete annelid worm Chaetopterus variopedatus. The molecule has been isolated from embryos at 15 h of development. It is a single peptide of about 200 kDa molecular weight, cross-reacting with antibodies against sea urchin DNA methyltransferase. The enzymatic properties of the molecule are similar to those of Dnmt1 methyltransferases isolated from other organisms, but with the peculiarity to be unable to make 'de novo' methylation on double stranded DNA.

Cooperative mechanism in the homodimeric myoglobin from Nassa mutabilis.

Oxygen binding and spectroscopic properties of the homodimeric myoglobin (Mb) from the prosobranchia sea snail Nassa mutabilis have been investigated. Oxygen equilibrium curves are pH-independent and cooperative with P50 = 5 +/- 1 mmHg and n approximately 1.5. Circular dichroism spectra of the oxygenated and deoxygenated form of N. mutabilis Mb are superimposable between 190 and 250 nm, suggesting a mechanism for cooperative ligand binding that does not involve changes in the alpha-helical content of the whole protein. The oxygen dissociation process is biphasic and pH-dependent, with different pKa values (=6.7 +/- 0.2 and 8.5 +/- 0.3) for the two phases. Moreover, the activation energy is essentially the same for both oxygen dissociation processes (Ea = 56.4 +/- 2.1 kJ/mol for the fast phase, and Ea = 53.8 +/- 1.9 kJ/mol for the slow phase), indicating that the rate difference for O2 dissociation between the diliganded and the monoliganded species is mostly dependent on a variation of the activation entropy. Ferrous nitrosylated N. mutabilis Mb shows, at alkaline and neutral pH, axial and rhombic X-band EPR signals, respectively, which display below pH 6 a three-hyperfine pattern typical of five-coordination. The results presented here suggest that in N.mutabilis Mb the kinetic control of cooperativity operates through a mechanism never observed before in other hemoproteins, which requires a ligand-linked large enhancement for the value of the oxygen association process in a molecule not undergoing changes in quaternary structure.

Organization and nucleotide sequence of the cluster of five histone genes in the polichaete worm Chaetopterus variopedatus: first record of a H1 histone gene in the phylum Annelida.

Histone genes were identified and their nucleotide sequences were determined in the polychaete marine worm Chaetopterus variopedatus. The genes are organized in about 390 clusters of 7.3 kbp. Each cluster contains one copy of the five histone genes. The H1 histone gene present in the clusters is the first ever isolated in the phylum Annelida. The cluster has the unique peculiarity that all genes contain both the replication-dependent and the replication-independent 3' mRNA termination signals. Despite the differences in cluster organization and transcription polarity of the individual histone genes between C. variopedatus and Platynereis dumerilii, the other annelid in which histone genes have been studied, phylogenetic analysis of the encoded amino acid sequences clearly groups together those two organisms in a tree in which the other studied worms find closely related positions on the same evolutionary branch.

Genome methylation of the marine annelid worm Chaetopterus variopedatus: methylation of a CpG in an expressed H1 histone gene.

Hydrolysis by methylation-dependent restriction enzymes shows that the genomic DNA of the polychaete annelid worm Chaetopterus variopedatus is methylated. Electrophoretic analyses of the digestion products indicate that the degree of methylation is lower in adult tissues than in sperm and embryonic DNA. 5-Methylcytosine was identified by HPLC, absorption spectroscopy and mass spectrometry analyses of free bases obtained by acid hydrolysis of the DNA. An average value of 1.6% methylated cytosines was determined in sperm DNA. Partial methylation was also found in an actively expressed H1 histone gene. This is the first time that genomic DNA methylation is demonstrated to occur in a worm.

In situ hybridization analysis of globin mRNAs in the primitive erythroid cells of the chick embryo.

The possibility that the minor embryonic chick hemoglobins might be present in a particular subgroup of primitive erythroid cells has been investigated by in situ hybridization. Probe to detect the mRNA for the alpha A globin chain of the minor embryonic hemoglobin was used, and the results of the hybridization were compared with those obtained using as probes the cDNAs for total globin mRNAs. All erythroid cells circulating in a 4-day-old chick embryo gave positive signals with both probes at an approximately constant ratio. This shows that all cells contain a similar assortment of hemoglobin types, excluding the possibility that a subgroup might contain the minor primitive hemoglobins exclusively. However, the cells are not homogeneous, since about 10% of them show a distinctly higher concentration of mRNA of all globin types.

Alteration of the proximal bond energy in the unliganded form of the homodimeric myoglobin from Nassa mutabilis. Kinetic and spectroscopic evidence.

CO binding kinetics to the homodimeric myoglobin (Mb) from Nassa mutabilis has been investigated between pH 1.9 and 7.0. Protonation of the proximal imidazole at low pH (less than or equal to 3.0) and the consequent cleavage of the HisF8NE2-Fe proximal bond brings about a approximately 20-fold increase of the second-order rate constant for CO binding. This process displays a pKa = 4.0 +/- 0.2, significantly higher than that observed in all other deoxygenated hemoproteins investigated up to now. Such a feature underlies a decreased energy for the HisF8NE2-Fe proximal bond in the unliganded form and it also appears supported by resonance Raman spectroscopy in the low frequency region of the Fe(II) deoxygenated hemoprotein. Further, the pH-rate profile of N. mutabilis Mb, like that of the homodimeric hemoglobin (Hb) from Scapharca inaequivalvis (Coletta, M., Boffi, A., Ascenzi, P., Brunori, M. and Chiancone, E. (1990) J. Biol. Chem. 265, 4828-4830), can be described only by assuming a concerted proton-linked transition with n = 1.8 +/- 0.1. Such a characteristic suggests, also on the basis of the amino acid sequence homology between N. mutabilis Mb and S. inaequivalvis Hb in the region forming the subunit interface, that the interaction mechanism is similar for the two homodimeric proteins, and drastically different Hb in the region forming the subunit interface, that the interaction mechanism is similar for the two homodimeric proteins, and drastically different from that operative in other hemoproteins.