Analysis of the replication mode of double minutes using the PCC technique combined with BrdUrd labeling

A cultured line of neuroblastoma cells (NB) was found to contain double minute chromosomes (DMs). DMs have been reported to be acentric and, therefore, to be segregated randomly into daughter cells without separating their sister elements. When NB cells were fused with Chinese hamster metaphase cells, prematurely condensed chromosomes (PCCs) were induced. DMs seen together with G₂ PCCs appeared to be closely paired, dot-like structures resembling DMs observable in metaphase cells. In contrast, DMs in G₁ cells showed a tendency to become single as the stage progressed so that the majority of DMs in late G₁ cells were actually no longer double. DMs in S-phase cells, however, again appeared double. These results clearly indicate why DMs are invariably double and never assume a quadruple configuration in metaphase cells in spite of their non-disjunctional segregation at anaphase. Such a characteristic mode of DM replication was further confirmed by a 5-bromo-2-deoxyuridine (BrdUrd) labeling experiment: when NB cells were exposed to BrdUrd for two successive rounds of DNA replication prior to PCC induction, half of the resulting single G₁ minutes as well as G₁ PCCs stained dark and the other half stained light after staining for sister chromatid differentiation.     

High-order structure of metaphase chromosomes: evidence for a multiple coiling model

When chromosome preparations made by the conventional air-drying method were processed with the OsO₄/TCH technique and examined by scanning electron microscopy (SEM), spiral structures in chromatids, which have been frequently observed to be present by light microscopy, were found to be composed of 30 nm fibres. In some portions these fibres appeared to be arranged in coils to form thicker fibres. When chromosome preparations were processed for SEM without air drying, chromosomes appeared to consist of fairly homogeneous thick fibrous structures measuring about 200 nm in diameter. In relatively condensed chromosomes, these 200 nm fibres appeared to be arranged perpendicular to the long axis of the chromatid. These findings suggest that chromatid spiral structures represent a regularly loosened state of the compactly spiralized 200 nm fibres which in turn consist of spiralized 30 nm fibres.     

Effects of Glyphosate on the Shikimate Pathway and Regulation of Phenylalanine Ammonia-lyase in Cryptomeria and Perilla Cell Suspension Cultures

Treatment of Cryptomeria and Perilla cell suspension cultureswith glyphosate resulted in a marked suppression of the formationof flavans and caffeic acid derivatives, respectively, whileit caused only a slight decline in the cell growth. In contrastwith 3-deoxy-D-arabino-heptulosonate (DAHP) synthase-Mn isozyme,DAHP synthase-Co isozyme from Cryptomeria and Perilla cellswas much more sensitive to inhibition by glyphosate. The additionof 1 to 2 mM glyphosate caused an accumulation of shikimateand quinate and a reduction of L-phenylalanine in both cellcultures. The inhibition of phenylalanine ammonia-lyase (PAL)activity by glyphosate was reversed by exogenously suppliedL-phenylalanine to near the control level. Cycloheximide andactinomycin D nullified the recovery by exogenous L-phenylalanineon PAL activity. L-Phenylalanine itself promoted PAL activityto some extent. No recovery of PAL activity in L--aminooxy-ß-phenylpropionate(L-AOPP)-treated cell cultures could be observed by the additionof L-phenylalanine. Therefore, L-AOPP seems to inhibit the formationof PAL, though it has been considered a competitive inhibitor. ³Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai 980, Japan. (Received October 28, 1985; Accepted March 13, 1986)     

Daphnia curvirostris Eylmann in Japanese high mountain waters (Cladocera: Daphniidae)

A species of Daphnia, Daphnia curvirostris Eylmann, found in high mountain lakes and ponds in central Japan is described. Although there were some differences in the shape of the male rostrum and the chromosome number between European populations as described by Johnson (1952) and Trentini (1980), and Japanese ones collected from high mountain waters, Japanese specimens had many characteristics similar to the taxon D. curvirostris of Europe.     

Primordial germ cells and lymphomyeloid system in the embryos of the Aleutian skate,Bathyraja aleutica

Organogenesis in embryos (1 and 5 mm-disk widths) of the Aleutian skate,Bathyraja aleutica was described histologicaily in complete serial paraffin sections. Special attention was paid to localization of primordial germ cells (PGC) and development of lymphoid tissues. The embryo of 1 mm-disk width was at the somite stage with no evidence of organogenesis. PGC, gathered in the genital ridge, were seen in the somatic mesodermal layer as well as in the mesenchyme between the endoderm and splanchnic mesoderm of the 1 mm-embryo. Most of the visceral organs were developing in the embryo of 5 mm-disk width. With respect to the development of immune system, two pairs of thymus anlagen, filled with numerous thymic lymphocytes, were recognized in the pharyngeal region. A small focus of numerous immature blood cells appeared to be an anlage of the spleen was found between the stomach and the liver.     

Gland formation induced in the allantoic and small-intestinal endoderm by the proventricular mesenchyme is not coupled with pepsinogen expression

Abstract. Allantoic and small-intestinal endoderms of chick and quail embryos were associated with the proventricular mesenchyme of chick embryos and then cultivated on chorioallantoic membrane. This resulted in the induction of complex glands, but the recombinates never produced embryo-specific pepsinogens; also, glandular cells developed a brush border, expressed sucrase antigen on their apical surface, and sometimes differentiated into goblet cells, thus indicating that both endoderms have the tendency to differentiate into an intestinal epithelium. In the recombinates composed of allantoic endoderm and proventricular mesenchyme, acid-protease activity was detected, but biochemical analysis revealed that this activity was not due topepsinogens. These results indicate that the gland formation induced in allantoic and small-intestinal endoderms by the proventricular mesenchyme is not accompanied by the expression of pepsinogens, suggesting that independent mechanisms are responsible for the morphogenesis and cyto chemical differentiation of the endoderm.     

Growth Regulation in Dwarf Barley Coleoptiles by the Minor Cell Wall Components, Galactose and Mannose

Sugar compositions of cell walls of dark-grown coleoptiles from12 barley strains, 11 of which were coleoptilar semi-dwarf strains,were analyzed on days 2 and 3 after germination. Major wallcomponents of the 12 strains were arabinose, xylose, and glucosein hemicellulose and cellulose; minor components were galactoseand mannose. The sugar content of each wall component per unit length wasnot correlated to any growth parameters calculated from a logisticequation simulating coleoptile growth, but the relative contentsof galactose and mannose in relation to the total wall sugarcontent was correlated to the growth rate on day 3 and the growthcontinuing period. These facts suggest that growth of these12 barley strains in the late growth stage is regulated by theminor wall components, galactose and mannose. ¹ Dedicated to the late Professor Joji Ashida. (Received October 12, 1982; Accepted January 12, 1983)     

The fine structure of the gill epithelium of a fresh-water flea,Daphnia magna (Crustacea: Phyllopoda) and changes associated with acclimation to various salinities

Two kinds of epithelial cells, dark and light types, are alternately arranged in the gill of Daphnia magna. The dark cell has numerous mitochondria and an elaborate tubular system containing two kinds of cytoplasmic tubules, small about 70 nm in diameter, and large about 130 nm in diameter. The former occur in bundles and seem to be smooth-surfaced endoplasmic reticulum. The latter, lined with a ridged surface coat and frequently open at the lateral and basal cell membrane, are regarded as extensions of the cell membrane. The atypical cell membrane of the dark cell is modified by repeated subunits of a cytoplasmic coat on the inner leaflet of the unit membrane. The light cell exhibits a high degree of basal infoldings of the cell membrane, which represent a magnification of the surface area of the cell. Large mitochondria between the infoldings often come into intimate association with the infolded cell membrane to form a regular array of parallel mitochondria interposed with the double cell membranes. The results suggest that at least the dark epithelial cells play an important role in the osmoregulation of this animal.     

R-Factor Mutant Capable of Specifying Hypersynthesis of Penicillinase

The physical characteristics of a mutant, R(M201-2), capable of conferring high and stable ampicillion resistance was analyzed. The R(M201-2) and its parent R-factor deoxyribonucleic acid (DNA) could be isolated as an extrachromosomal and covalently closed circular form. Their buoyant densities were both 1.712 g/cm(3), and their molecular weights were about 82 x 10(6) and 64 x 10(6), respectively, when measured by CsCl and sucrose density gradient analyses. The contour lengths by electron microscopy were 35.9 +/- 0.6 and 31.0 +/- 0.6 mum, respectively. By using the extracted R-factor DNA, the mutant and parent characters were transformable to another Escherichia coli strain. The mutant R factor showed an increased amount of DNA even after conjugal transfer to Proteus. An increase in the size of R-factor DNA was thus considered to be the cause of the high level of ampicillin resistance.