Identification of aggregation substances of Enterococcus faecalis cells after induction by sex pheromones

The sex pheromone system of Enterococcus faecalis is responsible for the clumping response of a plasmid carrying donor strain with a corresponding plasmid free recipient strain due to the production of sex pheromones by the recipient strain. The clumping response is mediated by a surface material (called aggregation substance) which is synthesized upon addition of sex pheromones to the cultures. Here we show that after induction a dense layer of hairlike structures is formed on the cell wall of the bacteria. These hairlike structures are responsible for the cell-cell contact which leads to the aggregation of cells. Formation of these structures was specific, only occurring after the addition of homologous sex pheromone.Abbreviations cAD1 sex pheromone specific for plasmid pAD1 - cPD1 sex pheromone specific for plasmid pPD1 - CW cell wall - pAD1 conjugative plasmid specifically transferred in the presence of cAD1 - pPD1 conjugative plasmid specifically transferred in the presence of cPD1 - PBS 10 mM Na-phosphate pH 7.5, 0.85% NaCl     

Comparative analysis of 18 sex pheromone plasmids fromEnterococcus faecalis: detection of a new insertion element on pPD1 and implications for the evolution of this plasmid family

A new IS element, IS1062, related to the enterococcal IS elements IS6770 and IS1252, was detected in the 3-terminus of the surface exclusion gene,sep1, of sex pheromone plasmid pPD1 inEnterococcus faecalis. pPD1-bearing cells lack the surface exclusion function, probably as a consequence of this insertion. Analysis of pAD1 and pPD1 sequences (7.5 kb and 2.7 kb, respectively) downstream of their aggregation substance genes revealed no similarity in these DNA regions. Detailed DNA/DNA hybridization studies using DNA probes specific for various pAD1-encoded genes needed for plasmid transfer indicated that the sex pheromone plasmids have evolved by repeated recombination and insertion of diverse transposable elements which presumably account for recent acquisition of antibiotic resistances.     

Steroid synthesizing cellular sites in the ovary of the domestic pigeon Columba livia (Gmelin): a histochemical study.

Synopsis The ovary of the domestic pigeon,Columba livia, has been assayed histochemically for the localization of ⁵-3-hydroxysteroid dehydrogenase (⁵-3-HSDH), 17-hydroxysteroid dehydrogenase (17-HSDH), 11-hydroxysteroid dehydrogenase (11-HSDH), glucose-6-phosphate dehydrogenase (G6P-DH) and NADH-diaphorase activities during different periods of the reproductive cycle. ⁵-3-HSDH, 17-HSDH, 11-HSDH, G6P-DH and NADH-diaphorase activity was found in the theca interna of growing, atretic and postovulatory follicles, the granulosa of ovulatory, atretic and postovulatory follicles, and interstitial gland cells during the pre-incubation and the laying periods. During the incubation and squab feeding periods only ⁵-3-HSDH, G6P-DH and NADH-diaphorase activities were observed in the above mentioned cells. The steroidogenic potential of atretic follicles depends upon the type of atresia a follicle undergoes.     

Sex pheromone plasmid pAD1-encoded surface exclusion protein of Enterococcus faecalis.

Summary During conjugative transfer of sex pheromone plasmids ofEnterococcus faecalis a so-called surface exclusion protein reduces the frequency with which these plasmids are transferred to cells already possessing the same plasmid. We report here the DNA sequence of a 3 .8 kb fragment of the sex pheromone plasmid pAD1 containing the structural genesea1 for surface exclusion protein and a small open reading frame (ORF) upstream ofsea1. Surface exclusion protein Seal was found to be highly homologous to the surface exclusion protein Sec10 encoded by the sex pheromone plasmid pCF10. Hybridization studies with DNA probes derived from the structural gene seal demonstrated that, with the exception of pAM373, all known sex pheromone plasmids carry a homologous gene. These studies also indicated that the genetic organization is similar in these plasmids, with the structural gene for surface exclusion protein being located 5 to that for aggregation substance.     

Production of active human interferon-β inE. coli I. Preferential production by lower culture temperature

Summary Unusually low culture temperature, such as 20°C, was shown to be preferable for the synthesis of active human interferon- (IFN-) inE. coli harboring a recombinant plasmid. TheE. coli cells cultured at 20°C gave 8.6-fold higher IFN- activity than those cultured at 37°C. However, almost the equal amounts of IFN- protein were accumulated in both cells cultured at 20°C and at temperature higher than 20°C, suggesting that IFN- might exist as an active form in the cells cultured at 20°C, while as a rather denatured form in the cells cultured at higher temperature.     

Immunochemical studies on barley seed storage proteins

The antigenic relationships between the prolamins of barley, rye and wheat have been studied by examining the specificity of an antibody to C hordein in a quantitative study using a laser nephelometer. The antibody reacts weakly with B hordein and strongly with 75-kdalton and 40-kdalton -secalins from rye and ₃ some -gliadins from wheat. Absorption experiments and immunodiffusion tests indicate that there are shared antigenic determinants for most of the prolamins. All the species with reacting prolamins belong to the sub-family Festucoideae of the Gramineae. The prolamins of maize, pearl millet and sorghum, species of the sub-family Panicoideae, do not react. The results confirm the known lack of homology between the prolamins of the two sub-families and also indicate the presence of relationships not yet established between C hordein, the 75-kdalton and 40-kdalton -secalins and also ₃ gliadin.Abbreviations HMW high molecular weight - PAGE polyacnylamide-gel electrophoresis - PBS phosphate-buffered saline - RLS relative light scattering - SDS sodlum dodecyl sulphate     

Effect of α-amanitine on puffing and intranuclear RNA synthesis in Chironomus salivary glands

Incubation of Chironomus salivary glands with -amanitine in concentrations from 1 to 10 /ml results in the suppression of puffing and chromosomal ³H-uridine incorporation after 30 to 60 min in 80% of the cells. Nucleolar ³H-uridine incorporation remains completely unaffected. Even 4 h after the injection of high doses of -amanitine into living larvae, nucleolar incorporation is still pronounced. The distribution of resistant cells within the salivary glands suggests that the uptake of -amanitine is subject to physiological restrictions.—A puff typically induced during in vitro incubation of salivary glands was found to be less sensitive to -amanitine than the Balbiani rings.     

Pheromone-induced phosphorylation of antennal proteins from insects

Protein kinase C inhibitors, such a calphostin C, abolish the transient nature of pheromone-induced rapid inositol 1,4,5-triphosphate (IP₃) responses, suggesting that pheromone signalling is terminated by phosphorylation of specific proteins. Challenging antennal preparations fromHeliothis virescens with species-specific pheromones in the presence of [³²P]--ATP led to a rapid, stimulus-dependent incorporation of³²P_i into antennal proteins. Pheromone-induced phosphorylation was completely abolished by a blockade of protein kinase C. Electrophoretic analysis revealed that upon stimulation with a pheromone blend two polypeptide bands were labelled; stimulation solely with the major compound (Z-11-hexadecenal) resulted in only a single labelled band. The data indicate that pheromones cause phosphorylation of specific antennal proteins which may be receptors for pheromones.Abbreviations ATP adenosine 5-triphosphate - DMSO dimethylsulphoxide - DPM disintigrations per minute - DTT dithiothreitol - EDTA ethylenediaminetetra-acetic acid - EGTA ethyleneglycol-bis(-aminoethyl ether)N,N,N,N-tetra-acetic acid - GTP guanosine 5-triphosphate - IP₃ inositol 1,4,5-trisphosphate - MOPS 3-(N-morpholino)propanesulphinic acid - P_i inorganic phosphate - PDBu phorbol-dibutyrate - SDS sodium dodecyl sulphate     

Amplification of plasmid copy number by thymidine kinase expression in Saccharomyces cerevisiae

Summary A 2 m circle-based chimaeric plasmid containing the yeast LEU2 and the Herpes Simplex Virus type 1 thymidine kinase (HSV-1 TK) genes was constructed. Transformants grown under selective conditions for the LEU2 gene harboured the plasmid at about 15 copies per cell whilst selection for the HSV-1 TK gene led to an increase to about 100 copies per cell. Furthermore, the plasmid copy number could be controlled by the stringency of selection for the TK gene, and the increase in TK gene dosage was reflected in an increase in intracellular thymidine kinase activity. The mitotic stability of the plasmid in high-copy and low-copy number cells was determined. High-copy number cells showed a greater mitotic stability. The relationship of TK expression to plasmid copy number may be useful for the isolation of plasmid copy number mutants in yeast and the control of heterologous gene expression.