Some properties of β-1,3-glucan hydrolyzing enzymes from the rotifer Brachionus plicatilis

We examined some characteristics of hydrolyticenzymes, especially -1,3-glucanase, to obtain theinformation of cell wall lytic enzymes forrotifers.Crude enzyme (ammonium sulfate fraction) of rotifershydrolyzed starch, -1,3-glucan, glycol chitinand CM-cellulose. Optimum pH for hydrolysis ofstarch and CM-cellulose was 6.5, and that for -1,3glucan and glycol chitin was pH 6.0. Pectic acid,xylan and agarose were not hydrolyzed at pH 3–10.-1,3 glucanase was purified about 73-fold from crudeenzyme by ion-exchange chromatography and gelfiltration. Optimum pH and temperature of the enzymewere 6 and 60 °C, respectively. The molecular weight ofthe enzyme was estimated about 260 kDa by gelfiltration. The enzyme was inhibited byHgCl₂ and MnCl_{_2}.     

Measurement of Stomatal Aperture by Digital Image Processing

We developed a new digital image processing technique for exactlymeasuring the degree of stomatal opening, that is, the ratioof the width to the maximum length of a stomatal pore, and thepore area. We applied this technique to evaluate responses toSO₂ of neighboring stomata in a small region of an intact attachedleaf, with the following results:

1. The pore region could be exactly extracted even when the originaldigital image was of poor quality. The standard errors in theevaluation of the pore area, the pore length, and the pore widthwere 10 µm², 0.3 µm and 0.3 µm, respectively.
2. A wide variety of responses to SO₂ was found among neighboringstomata in a small region of an attached leaf. The differenceswere especially remarkable before water-soaking and wilting.When these began, k₁ showed a local maximum value, and thenall the stomata began to close. These results suggest that stomatalresponses of intact growing plants to air pollutants shouldbe examined from the standpoint of not only the average movementof many stomata but also the movement of each stoma and thatthe interrelationship among movements of neighboring stomatashould be investigated to clarify the mechanism of stomatalresponses to air pollutants.

(Received June 4, 1984; Accepted August 23, 1984)     

Studies on antiviral glycosides. II. Mode of action for virucidal effects on Sendai virus

Treatment of Sendai virus with $\text{p-(sec-}\text{butyl)-phenyl-6-chloro-6-deoxy-β-}\text{d}\text{-glucopyranoside}$, followed by freezing and thawing resulted in a loss of hemolytic and cell fusion activities as well as infectivity without affecting hemagglutinating and neuraminidase activities. The anti-hemolytic activity of this compound was reversed by the addition of phosphatidyl choline to the virus samples. $\text{p-}\text{Azidophenyl-6-chloro-6-deoxy-β-}\text{d}\text{-[}{}^3\text{H]glucopyranoside}$ was successfully used for photoaffinity labeling of a specific virion site, and we confirmed the affected site of the glucoside to be the lipid components in the viral envelopes.     

Ultrastructure of intracytoplasmic membranes of Methanomonas margaritae cells grown under different conditions

The development of intracytoplasmic membranes of Methanomonas margaritae cells grown under different culture conditions was studied. Growth on methane was strongly accelerated by the addition of copper ions. Acceleration by copper, however, was not observed in the case of growth on methanol. Cells grown on methane with copper possessed intracytoplasmic membranes along the cell periphery. When the organism was grown in a medium lacking copper, intracytoplasmic membranes appeared as large vesicles surrounded by a unit membrane at the periphery of the cell. The vesicles originated from paired membranes due to the absence of copper in the medium. Cells grown on methanol with or without copper possessed a number of vesicles of different sizes arranged in a chain along the cell periphery. The possible relationship between membrane arrangement and methane oxidation is discussed.     

Cloning of Bacillus subtilis leucina A, B and C genes with Escherichia coli plasmids and expression of the leuC gene in E. coli.

Summary The leucine genes of Bacillus subtilis have been cloned directly from the chromosomal DNA into Escherichia coli leuB cells by selection for the Leu⁺ phenotype using RSF2124 as a vector plasmid. The hybrid plasmid designated RSF2124-B·leu contained a 4.2 megadalton fragment derived from B. subtilis DNA, including the leu genes. The fragment had one site susceptible to EcoRI^* and another site susceptible to BamNI endonuclease. Among the three fragments produced by EcoRI^* and BamNI endonucleases, the 1.2 megadalton fragment had the ability to transform B. subtilis leuA, leuB and leuC auxotrophs to leu ⁺. However, B. subtilis ilvB and ilvC auxotrophs were not rescued even by the whole 4.2 megadalton fragment present in the hybrid plasmid. -Isopropylmalate dehydrogenase (leuB gene product) activity found in E. coli cells containing the hybrid plasmid was about 60% of that in E. coli wild type cells, despite the high copy number (7.8) of the plasmid per chromosome observed.     

Changes in structural organization of surface membrane during erythrocyte maturation

The effect of concanavalin A and its succinylated derivative on cell agglutination and potassium compartmentation of mature and immature erythrocytes was observed. The binding of tetravalent concanavalin A to the surface glycoproteins of rabbit erythrocytes leads to a change in the properties of the surface membrane, which results in an induction of cell agglutination and concomitant release of potassium from the cells. Both of the phenomena induced by concanavalin A are temperature dependent, and observed at above 15°C.Divalent succinylated concanavalin A, lacking the inducing activity of surface glycoprotein cross-linking into patches and caps, caused neither cell agglutination nor change in the potassium compartmentation of erythrocytes and reticulocytes.In the case of immature reticulocytes, however, remarkable agglutination of the cells was induced without a change in the potassium compartmentation after treatment with tetravalent concanavalin A.It is suggested that changes in the molecular organization of the surface membrane occur in which potassium compartmentation of the reticulocytes becomes more susceptible to surface glycoprotein cross-linking during cellular maturation.     

Methylamine facilitates demonstration of specific uptake of diphtheria toxin by CHO cell and toxin-resistant CHO cell mutants

We have measured the specific uptake of ¹²⁵I-labelled diphtheria toxin in the presence of methylamine by a number of cell lines with different sensitivities to diphtheria toxin. The results show a strong correlation between the toxin sensitivities of the cell lines and the amount of specific uptake. The specific association of labelled toxin with cells was clearly demonstrated even with CHO cells, a cell line with relatively low sensitivity. Thus, CHO cell mutants that are resistant to diphtheria toxin could be classified as toxin-binding or non-binding cells by this method.     

Gene conversion in the Escherichia coli RecF pathway: a successive half crossing-over model.

Summary Gene conversion - apparently non-reciprocal transfer of sequence information between homologous DNA sequences - has been reported in various organisms. Frequent association of gene conversion with reciprocal exchange (crossing-over) of the flanking sequences in meiosis has formed the basis of the current view that gene conversion reflects events at the site of interaction during homologous recombination. In order to analyze mechanisms of gene conversion and homologous recombination in an Escherichia coli strain with an active RecF pathway (recBC sbcBC), we first established in cells of this strain a plasmid carrying two mutant neo genes, each deleted for a different gene segment, in inverted orientation. We then selected kanamycin-resistant plasmids that had reconstituted an intact neo ⁺ gene by homologous recombination. We found that all the neo ⁺ plasmids from these clones belonged to the gene-conversion type in the sense that they carried one neo ⁺ gene and retained one of the mutant neo genes. This apparent gene conversion was, however, only very rarely accompanied by apparent crossing-over of the flanking sequences. This is in contrast to the case in a rec ⁺ strain. or in a strain with an active RecE pathway (recBC sbcA). Our further analyses, especially comparisons with apparent gene conversion in the rec ⁺ strain, led us to propose a mechanism for this biased gene conversion. This successive half crossing-over model proposes that the elementary recombinational process is half crossing;-over in the sense that it generates only one recombinant DNA duplex molecule, and leaves one or two free end(s), out of two parental DNA duplexes. The resulting free end is, the model assumes, recombinogenic and frequently engages in a second round of half crossing-over with the recombinant duplex. The products resulting from such interaction involving two molecules of the plasmid would be classified as belonging to the gene-conversion type without crossing-over. We constructed a dimeric molecule that mimics the intermediate form hypothesized in this model and introduced it into cells. Biased gene conversion products were obtained in this reconstruction experiment. The half crossing-over mechanism can also explain formation of huge linear multimers of bacterial plasmids, the nature of transcribable recombination products in bacterial conjugation, chromosomal gene conversion not accompanied by flanking exchange (like that in yeast mating-type switching), and antigenic variation in microorganisms.