Pulsed-field fingerprinting of listeriae: identification of genomic divisions for Listeria monocytogenes and their correlation with serovar.

Clamped homogeneous electric field (CHEF) electrophoresis was optimized for genomic analyses of Listeria monocytogenes. Various human, animal, food, and environmental isolates, as well as strains representing other Listeria species, were separately digested with rarely cutting endonucleases. Of 176 L. monocytogenes strains analyzed, the enzymes AscI and ApaI established 63 and 72 unique restriction endonuclease digestion profiles (REDP), respectively. The 22 non-L. monocytogenes strains exhibited 18 AscI and 19 ApaI unique REDP. Statistical analyses of REDP information using the Dice coincidence index and principal component analysis revealed two distinct genomic divisions of L. monocytogenes that also correlated with the flagellar (H) antigen type: division I contained serovar 1/2a, 1/2c, 3a, and 3c stains and division II contained serovar 1/2b, 3b, 4b, 4d, and 4e strains. Division I isolates digested with ApaI were further grouped into cluster IA (serovar 1/2c and 3c) and cluster IB (serovar 1/2a and 3a) strains. Likewise, division II isolates digested with ApaI were further grouped into cluster IIA (serovar 1/2b and 3b) and cluster IIB (serovar 4b, 4d, and 4e) strains. These data indicate that genotypic data generated by CHEF can be directly related to phenotypic data generated by serotyping for establishing the overall relatedness of isolates. Moreover, these data further substantiate that CHEF analysis is a reproducible and highly discriminating method for characterizing L. monocytogenes strains at the molecular level.     

Bone sialoprotein mRNA expression and ultrastructural localization in fetal porcine calvarial bone: comparisons with osteopontin

Summary Bone sialoprotein (BSP) and osteopontin (OPN) are two major non-collagenous proteins in bone that have similar biochemical properties and can mediate cell attachment through an RGD (Arg-Gly-Asp) motif that recognizes the vitronectin receptor. To facilitate evaluations of the biological functions of BSP and OPN in bone formation, affinity-purified rabbit polyclonal antibodies against porcine BSP and OPN were used, together with a high-resolution protein A-gold immunocytochemical technique to reveal the ultrastructural localization of these proteins in undermineralized sections of 50-day fetal porcine calvarial bone. In addition,³⁵S-labelled antisense riboprobes were prepared to demonstrate the cellular expression of BSP and OPN in the same tissues usingin situ hybridization. Immunolocalization for both BSP and OPN revealed the highest density of gold particles associated with electron-dense organic material found at the mineralization front and in ‘cement lines’. Labelling was also observed in the mineralized matrix over electron-dense material between collagen fibrils. In the osteoid of newly-formed bone, immunogold labelling for BSP and OPN was associated with loci of mineralization, which were often characterized by feathery clusters of fine needle-like crystals. Results ofin situ hybridization on the same tissues demonstrated that BSP mRNA expression was restricted to differentiated osteoblasts with particularly strong signals evident at sites ofde novo bone formation. More moderate expression of BSP was observed in ‘older’ osteoblasts and in some of the newly-entrapped osteocytes. Although expression of OPN mRNA was also observed in osteoblasts and osteocytes, the level of hybridization was similar for most bone cells and not markedly stronger than the signal observed in some stromal cells. While it is evident from these and other studies that both BSP and OPN are associated with bone formation, the differences observed in cellular expression indicate distinct roles for these proteins in bone formation.     

<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 effect of ferredoxin(BED) overexpression on benzene dioxygenase activity in Pseudomonas putida ML2.

The benzene dioxygenase from Pseudomonas putida ML2 is a multicomponent complex comprising a flavoprotein reductase, a ferredoxin, and a terminal iron-sulfur protein (ISP). The catalytic activity of the isolated complex shows a nonlinear relationship with protein concentration in cell extracts, with the limiting factor for activity in vitro being ferredoxin(BED). The relative levels of the three components were analyzed by using 125I-labelled antibodies, and the functional molar ratio of ISP(BED), ferredoxin(BED), and reductase(BED) was shown to be 1:0.9:0.8, respectively. The concentration of ferredoxin(BED) was confirmed by quantitative electron paramagnetic resonance spectroscopy of the 2Fe-2S centers in ferredoxin(BED) and ISP(BED) of whole cells. These results demonstrate that the ferredoxin(BED) component is a limiting factor in dioxygenase activity in vitro. To determine if it is a limiting factor in vivo, a plasmid (pJRM606) overproducing ferredoxin(BED) was introduced into P. putida ML2. The benzene dioxygenase activity of this strain, measured in cell extracts, was fivefold greater than in the wild type, and the activity was linear with protein concentration in cell extracts above 2 mg/ml. Western blotting (immunoblotting) and electron paramagnetic resonance spectroscopic analysis confirmed an elevated level of ferredoxin(BED) protein and active redox centers in the recombinant strain. However, in these cells, the increased level of ferredoxin(BED) had no effect on the overall rate of benzene oxidation by whole cells. Thus, we conclude that ferredoxin(BED) is not limiting at the high intracellular concentration (0.48 mM) found in cells.     

Phylogeny and physiology of Drosophila opsins

Phylogenetic and physiological methods were used to study the evolution of the opsin gene family in Drosophila. A phylogeny based on DNA sequences from 13 opsin genes including representatives from the two major subgenera of Drosophila shows six major, well-supported clades: The blue opsin clade includes all of the Rhl and Rh2 genes and is separated into two distinct subclades of Rhl sequences and Rh2 sequences; the ultraviolet opsin clade includes all Rh3 and Rh4 genes and bifurcates into separate Rh3 and Rh4 clades. The duplications that generated this gene family most likely took place before the evolution of the subgenera Drosophila and Sophophora and their component species groups. Numerous changes have occurred in these genes since the duplications, including the loss and/or gain of introns in the different genes and even within the Rhl and Rh4 clades. Despite these changes, the spectral sensitivity of each of the opsins has remained remarkably fixed in a sample of four species representing two species groups in each of the two subgenera. All of the strains that were investigated had R1-6 (Rhl) spectral sensitivity curves that peaked at or near 480 nm, R7 (Rh3 and Rh4) peaks in the ultraviolet range, and ocellar (Rh2) peaks near 420 nm. Each of the four gene clades on the phylogeny exhibits very conservative patterns of amino acid replacement in domains of the protein thought to influence spectral sen sitivity, reflecting strong constraints on the spectrum of light visible to Drosophila.     

Taxonomy, biology and abundance of icefishes, or noodlefishes (Salangidae), in the Yellow River estuary of the Bohai Sea, China

Four species of icefishes were sampled in the Yellow River estuary of the Bohai Sea from April 1984 to April 1985 and from February 1988 to June 1989. Morphological analysis showed that they belonged to three subfamilies of Salangidae: one species from Protosalangicae, Protosalanx chinensis ; two species from Salanginae, Salanx ariakensis and Salanx prognathus ; and one species, Neosalanx andersoni , from Salangichthyinae, a new subfamily. External morphological characteristics, population composition, growth, maturity, food habits, longevity, fecundity, abundance and distribution of these icefishes are described. Based on these analyses, a clear outline of taxonomy, zoogeography aod biology of icefishes in the Yellow River estuary of the Bohai Sea was established.