Phylogenetic relationship of the green alga Nanochlorum eukaryotum deduced from its chloroplast rRNA sequences.

The marine green coccoidal alga Nanochlorum eukaryotum (N.e.) is of small size with an average diameter of 1.5 microns. It is characterized by primitive-appearing biochemical and morphological properties, which are considerably different from those of other green algae. Thus, it has been proposed that N.e. may be an early developed algal form. To prove this hypothesis, DNA of N.e. was isolated by a phenol extraction procedure, and the chloroplast DNA separated by preparative CsCl density-gradient centrifugation. The kinetic complexity of the nuclear and of the chloroplast DNA was evaluated by reassociation kinetics to 3 x 10(7) bp and 9 x 10(4) bp, respectively. Several chloroplast genes, including the rRNA genes, were cloned on distinct fragments. The order of the rRNA genes corresponds to the common prokaryotic pattern. The 16S rRNA gene comprises 1,548 bases and is separated from the 23S rRNA gene with its 2,920 bases by a short spacer of 460 bases, which also includes the tRNA(Ile) and tRNA(Ala) genes. The 5S rRNA gene has not been found; it must start further than 500 bases downstream from the 3'-end of the 23S rRNA gene. From the chloroplast rRNA sequences, we have deduced secondary structures of the 16S and 23S rRNAs, which are in agreement with standard models. The rRNA sequences were aligned with corresponding chloroplast sequences; phylogenetic relationships were calculated by several methods. From these calculations, we conclude that N.e. is most closely related to Chlorella vulgaris. Therefore, N.e. does not represent an early developed algal species; the primitive-appearing morphological and biochemical characteristics of N.e. must rather be explained by secondary losses.

Ubiquitin and ubiquitination in cells from the marine sponge Geodia cydonium.

Marine sponges, e.g. Geodia cydonium, have been intensively used to investigate the biochemical and molecular biological basis of cell-cell- and cell-matrix adhesion. It has been shown that a family of galactose-specific lectins, which are present in the extracellular space of G. cydonium, is a main component involved in cell-matrix adhesion in the sponge system. In the present study it is outlined that the purified 16-kDa lectin-1 binds to a 67-kDa membrane-associated protein. This lectin-binding protein undergoes mono- and diubiquitination after incubation of dissociated sponge cells with the homologous aggregation factor (AF), a molecule involved in cell-cell adhesion. The gene coding for polyubiquitin was characterized and found to be composed of three tandem repeat building blocks. Northern analysis indicated the presence of only one type of ubiquitin-specific mRNA (1.65 kb). The level of this transcript increased by 10-fold after incubation of the dissociated cells with AF for 8 h; in contrast, lectin-1 caused only a small effect on the steady-state level of ubiquitin mRNA. These data indicate that the expression of the polyubiquitin gene is directly or indirectly regulated by the AF and suggest that ubiquitination might be a process which controls the function of the membrane-associated lectin-binding protein during matrix-cell adhesion.

Characterisation of cryptic plasmids in clinical isolates of Bacteroides fragilis.

A screening method for plasmids in the fragilis group of Bacteroides spp. was developed, taking account of the lysozyme resistance of these species; 26 strains, 24 of them B. fragilis, were investigated by this method. Eleven strains contained plasmids and up to three different plasmids were found in individual strains. The plasmids belonged to five different size classes of mol. wt (10(6] 2.8, 3.5, 3.6, 4.2 and 19. Plasmids of equal size showed homology; no homology was found between plasmids of different sizes. Plasmids of equal size showed identical restriction patterns with 17 restriction endonucleases. Restriction maps were constructed for the five classes of plasmid.

Equilibrium binding constants of C3DP, a nucleic acid-binding protein isolated from human serum.

The malignancy-associated DNA-binding protein C3DP has been isolated from normal human serum and identified by dodecyl sulfate/polyacrylamide gradient gel electrophoresis and immunodiffusion. The binding of purified C3DP to DNA and RNA has been investigated by density gradient centrifugation. The equilibrium binding constant K of C3DP binding to native human DNA was determined to be K = (2.9 +/- 0.5) x 10(5) M-1. From this value we conclude that C3DP binds non-specifically with regard to the base sequence of the DNA. K was found to be in the same order of magnitude when measured with single-stranded or double-stranded DNA of varying G + C-content, and with RNA. It varied only slightly with the pH or ionic strength of the medium, indicating that only one Na ion is released from DNA when one molecule of C3DP is bound. We conclude that ionic interactions cannot account for the stability of the C3DP-DNA complex and that therefore hydrogen bonds and hydrophobic interactions are likely to be formed.

Malignancy-associated DNA-binding protein C3DP from human serum. Further characterization and purification of C3DP retaining its DNA-binding affinity.

C3DP, a malignancy-associated DNA-binding protein from human serum[1], was purified to homogeneity without loss of its DNA-binding affinity. For this purpose normal human serum was submitted to affinity chromatography on Con A-Sepharose and DNA-cellulose and to preparative polyacrylamide gel electrophoresis. The purified C3DP was identified by immunodiffusion and sodium dodecylsulfate polyacrylamide gel electrophoresis and it was shown to bind to DNA by DNA-cellulose chromatography. The isoelectric point of C3DP was determined to 4.9 by isoelectric focusing.