Database on the structure of small ribosomal subunit RNA.

The database on small ribosomal subunit RNA structure contains (June 1994) 2824 nucleotide sequences. All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which in turn is corroborated by the observation of compensating substitutions in the alignment. The complete database is made available to the scientific community through anonymous ftp on our server in Antwerp. A special effort was made to improve electronic retrieval and a program is supplied that allows to create different file formats. The database can also be obtained from the EMBL nucleotide sequence library.     

Restriction generated oligonucleotides utilizing the two base recognition endonuclease CviJI*.

The conversion of an anonymous DNA sample into numerous oligonucleotides is enzymatically feasible using an unusual restriction endonuclease, CviJI. Depending on reaction conditions, CviJI is capable of digesting DNA at a two or three base recognition sequence. CviJI normally cleaves RGCY sites between the G and C to leave blunt ends. Under 'relaxed' conditions CviJI* cleaves RGCY, and RGCR/YGCY, but not YGCR sites. In theory, CviJI* restriction of pUC19 (2686 bp) should produce 157 fragments, 75% of which are smaller than 20 bp. Instead, 96% of the CviJI* fragments were 18-56 bp long and none of the fragments were smaller than 18 bp. Thermal denaturation of these fragments generates sequence specific oligonucleotides homologous for the cognate template. The enzymatic conversion of anonymous DNA into sequence specific oligomers has implications for several conventional and novel molecular biology procedures.     

Molecular characterization of de novo secondary trisomy 13.

Unbalanced Robertsonian translocations are a significant cause of mental retardation and fetal wastage. The majority of homologous rearrangements of chromosome 21 in Down syndrome have been shown to be isochromosomes. Aside from chromosome 21, very little is known about other acrocentric homologous rearrangements. In this study, four cases of de novo secondary trisomy 13 are presented. FISH using alpha-satellite sequences, rDNA, and a pTRI-6 satellite I sequence specific to the short arm of chromosome 13 showed all four rearrangements to be dicentric and apparently devoid of ribosomal genes. Three of four rearrangements retained the pTRI-6 satellite I sequence. Case 1 was the exception, showing a deletion of this sequence in the rearrangement, although both parental chromosomes 13 had strong positive hybridization signals. Eleven microsatellite markers from chromosome 13 were also used to characterize the rearrangements. Of the four possible outcomes, one maternal Robertsonian translocation, two paternal isochromosomes, and one maternal isochromosome were observed. A double recombination was observed in the maternally derived rob(13q13q). No recombination events were detected in any isochromosome. The parental origins and molecular chromosomal structure of these cases are compared with previous studies of de novo acrocentric rearrangements.     

<Emphasis Type="Italic">Alona alsafadii</Emphasis> n. sp. from Yemen,a primitive,groundwater-dwelling member of the <Emphasis Type="Italic">A. karua</Emphasis>-group

The effect of algae on the production of musty-smelling compounds by actinomycetes was studied. Streptomyces spp., causing intensive musty odor, were isolated from hypertrophic Lake Kasumigaura and cultured in association with algae from the same lake. Isolate E and I effectively utilized the cyanobacteria, Microcystis aeruginosa and Anabaena spiroides, and the diatom, Synedra acus, as a carbon source and produced a musty-smelling 2-methylisoborneol in the shaken sediment cultures. High populations of algae and actinomycetes, and aerobic condition in the sediment seem responsible for the occurrence of musty odor in Lake Kasumigaura.     

The fas operon of Rhodococcus fascians encodes new genes required for efficient fasciation of host plants.

Three virulence loci (fas, att, and hyp) of Rhodococcus fascians D188 have been identified on a 200-kb conjugative linear plasmid (pFiD188). The fas locus was delimited to a 6.5-kb DNA fragment by insertion mutagenesis, single homologous disruptive recombination, and in trans complementation of different avirulent insertion mutants. The locus is arranged as a large operon containing six open reading frames whose expression is specifically induced during the interaction with host plants. One predicted protein is homologous to P-450 cytochromes from actinomycetes. The putative ferredoxin component is of a novel type containing additional domains homologous to transketolases from chemoautotrophic, photosynthetic, and methylotrophic microorganisms. Genetic analysis revealed that fas encodes, in addition to the previously identified ipt, at least two new genes that are involved in fasciation development, one of which is only required on older tobacco plants.     

Determination of the gene sequence and the molecular structure of the enterococcal peptide antibiotic AS-48.

The structural gene of the enterococcal peptide antibiotic AS-48 (as-48) has been identified and cloned by using two degenerate 17-mer DNA oligonucleotides on the basis of the amino acid sequences of two peptides obtained by digestion of the antibiotic with Glu-C endoproteinase. That as-48 gene codes for a 105-amino-acid prepeptide, giving rise to a 70-amino-acid mature protein. Comparative analysis demonstrated that the 16-amino-acid sequence of one of the AS-48 Glu-C peptides, designated V8-5, was composed of a 12-amino-acid sequence corresponding to the C-terminal end sequence (from isoleucine +59 to tryptophan +70 [I+59 to W+70]) of the prepeptide and terminated in four residues forming the N terminus (M+1 to E+4) of a putative AS-48 propeptide. These data, combined with the characteristics of the gene sequence, strongly suggested that the antibiotic peptide was a 70-residue cyclic molecule. We propose that the AS-48 translated primary product is very likely submitted to a posttranslational modification during secretion (i) by an atypical or a typical signal peptidase that cleaves off a 35-residue or shorter signal peptide, respectively, from the prepeptide molecule and (ii) by the linkage of the methionine residue (M+1) to the C-terminal tryptophan residue (W+70) to obtain the cyclic peptide (a tail-head linkage).     

Root colonization and systemic spreading of Azoarcus sp. strain BH72 in grasses.

The invasive properties of Azoarcus sp. strain BH72, an endorhizospheric isolate of Kallar grass, on gnotobiotically grown seedlings of Oryza sativa IR36 and Leptochloa fusca (L.) Kunth were studied. Additionally, Azoarcus spp. were localized in roots of field-grown Kallar grass. To facilitate localization and to assure identity of bacteria, genetically engineered microorganisms expressing beta-glucuronidase were also used as inocula. beta-Glucuronidase staining indicated that the apical region of the root behind the meristem was the most intensively colonized. Light and electron microscopy showed that strain BH72 penetrated the rhizoplane preferentially in the zones of elongation and differentiation and colonized the root interior inter- and intracellularly. In addition to the root cortex, stelar tissue was also colonized; bacteria were found in the xylem. No evidence was obtained that Azoarcus spp. could reside in living plant cells; rather, plant cells were apparently destroyed after bacteria had penetrated the cell wall. A common pathogenicity test on tobacco leaves provided no evidence that representative strains of Azoarcus spp. are phytopathogenic. Compared with the control, inoculation with strain BH72 significantly promoted growth of rice seedlings. This effect was reversed when the plant medium was supplemented with malate (0.2 g/liter). N2 fixation was apparently not involved, because the same response was obtained with a nifK mutant of strain BH72, which has a Nif- phenotype. Also, Western blot (immunoblot) analysis of protein extracts from rice seedlings gave no indication that nitrogenase was present. PCR and Western immunoblotting, using primers specific for eubacteria and antibodies recognizing type-specific antigens, respectively, indicated that strain BH72 could colonize rice plants systemically, probably mediated by longitudinal spreading through vessels.     

Co-localization of cytokinins with proteins related to cell proliferation in developing somatic embryos of Dactylis glomerata L.

The present report provides evidence for co-localization ofcytokinins with cell proliferation-associated nuclear proteins.Somatic embryos of Dactylis glomerata in two stages of developmentare used as a model system comprising both proliferating andinitially differentiated cells. Cytokinins are localized usingantibodies with marked specificity against isopentenyladenine/adenosine(2iP/2iPA) or zeatin/ riboside (Z/ZR). The proliferation-associatednuclear antigen, mitotin, is analysed using a specific monoclonalantibody. The nuclear protein BM28, required for the onset ofDNA replication and for cell division, is identified by an affinity-purifiedpolyclonal antibody. Using double immunofiuorescence labellingwith the antibodies against cytokinins and against each of thenuclear proteins, immunoreaction is observed generally in thesame nuclei of almost all cells in globular embryos and in thenuclei of cells in meristematic areas of the more developedembryos. Only small numbers of individual nuclei positive forboth type of antibodies were found in the surrounding vacuolatedparenchymatous cells. The occurrence of plant antigens homologousto BM28 and mitotin is confirmed by immunoblotting assay. InSDS-PAGE blots the anti-BM28 antibody reacts with a proteinof 58 kDa. The anti-mitotin antibody recognizes several (160,140, 125, 93, and 80 kDa) polypeptides. The data showing nuclearco-localization of cytokinins and proteins with a suggestedrole in the onset of DNA synthesis and in cell division providea new base for further study on the mode of action of cytokininsin cell cycle regulation. Key words: Immunolocalization, cytokinins, nuclear proteins, mitotin, BM28, cell proliferation, somatic embryo(s), Dactylis glomerata