Existence of a Common Glycoprotein Homologous to S-Glycoproteins in Two Self-incompatible Homozygotes of Brassica campestris

S-Glycoproteins (S-locus-specific glycoproteins) in Brassicaspecies are present only in stigmas and thought to play an importantrole in self-incompatibility system. The stigma extract containsalso several other glycoproteins reacting with the antiserumto S-glycoproteins, among which some glycoproteins from S₈S₈-and S₉S₉-homozygotes have the same pI value. Both of the glycoproteinswhich were tentatively termed NS₈- and NS₈S₉-glycoproteins,respectively, were isolated and analyzed. Those were revealedto be identical. Its amino acid sequence was homologous withthe S-glycoproteins in Brassica species. The NS-glycoproteinswere expressed at the same time and only in stigma as S-glycoproteins. (Received July 19, 1988; Accepted September 7, 1988)     

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.     

Activated c-raf gene in a rat hepatocellular carcinoma induced by 2-amino-3-methylimidazo[4,5-f]quinoline

A rat hepatocellular carcinoma, IQ7, induced by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) gave two transformants of NIH 3T3 cells on DNA mediated gene transfer. One of these transformants was examined further and secondary and tertiary transformants were obtained. The secondary transformant was tumorigenic in nude mice. The activated oncogene in this primary transformant was identified as rat c-raf by Southern blot analysis.     

Location of polar substituents and fatty acyl chains on lipid A precursors from a 3-deoxy-D-manno-octulosonic acid-deficient mutant of Salmonella typhimurium. Studies by 1H, 13C, and 31P nuclear magnetic resonance

Eight anionic disaccharide precursors of lipid A accumulate at 42 degrees C in 3-deoxy-D-manno-octulosonic acid-deficient temperature-sensitive mutants of Salmonella typhimurium. These compounds comprise a series of lipids based on the minimal structure, O-[2-amino-2-deoxy-N2,O3-bis(3-hydroxytetradecanoyl)-beta-D-glucopyranos yl] -(1----6)-2-amino-2-deoxy-N2, O3-bis(3-hydroxytetradecanoyl)-alpha-D-glucopyranose 1,4'- bisphosphate (designated lipid IVA) that differ from each other by the presence of an additional phosphoethanolamine moiety (IIIA), or an aminodeoxypentose moiety (IIA), or both (IA). A homologous set of metabolites is further derivatized with a palmitoyl function; these are designated IVB, IIIB, IIB, and IB (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16088). The attachment of the palmitoyl moiety, known to be on the reducing terminal GlcN residue by mass spectrometry, was determined to be O-beta of the N2-linked beta-hydroxymyristoyl group of that residue of IVB by 13C NMR and two-dimensional 1H chemical shift correlation spectroscopy experiments. 31P NMR indicated the presence of diphosphodiester moieties in IIIA, IIIB, and IA and monophosphodiester moieties in IIA and IA. Selective 1H decoupling of the 31P spectrum of IIIA demonstrated that the O-diphosphoethanolamine moiety is attached to the O4' position in IIIA. On the basis of the observed 31P chemical shifts it was concluded that the aminodeoxypentose is located at position 1 in IIA and IA, while diphosphoethanolamine is most likely located at O-4' in IA and IIIB, as in IIIA.     

Effects of incubation in an atmosphere of 20% CO2 in air on the syrian hamster embryo clonal transformation assay

Summary An atmosphere containing 10% CO₂ has been generally accepted as optimal for the growth of Syrian hamster embryo cells in a clonal transformation assay. Data presented in this paper show that 10% CO₂ may not be the optimum environment for this assay. Using 10 or 20% (analytically measured) CO₂ in air (1 atm pressure), hamster embryo cell pools were examined for clonal growth characteristics and transformability using five known carcinogens and a single noncarcinogenic compound. At 10% CO₂, only 2 of 11 pools weee transformed by the five carcinogens but not by the noncarcinogen. At 20% CO₂, six of seven pools were transformed by the five carcinogens and not by the noncarcinogen. Further, the transformation frequencies were found to be greater in cultures incubated in an atmosphere consisting of 20% CO₂ in air. The data also show that 20% CO₂ increased the cloning efficiency of these cells. A comparison of the 10 and 20% CO₂ data to results reported from other conflicting interlaboratory results with this assay system may be due, in part, to variations of CO₂ concentrations. In some instances, the CO₂ levels indicated by incubator flow meters vary considerably from analytically determined CO₂ values. To prevent these CO₂ discrepancies and their resultant effects on transformation and cloning efficiency, methods for monitoring the CO₂ environment other than flow meters are recommended. The observation of increased cloning efficiencies and transformation rates strongly suggests that culture incubation at 20% CO₂ is a preferred environment for the conduct of this assay.     

Biological and physicochemical characterization of recombinant human erythropoietins fractionated by Mono Q column chromatography and their modification with sialytransferase

Ten erythropoietin (EPO) fractions differing in sialic acid content, ranging from 9.5 to 13.8 mol mol^–1 of EPO, were obtained from baby hamster kidney cell-derived recombinant human EPO by Mono Q column chromatography. The mean pI values of the EPO fractions determined by IEF-gel electrophoresis systematically shifted from 4.11 to 3.31, coinciding with the sialic acid content, without a change in the constitution of asialo N-linked oligosaccharides of each fraction. Although a linear relationship between thein vivo bioactivity and the sialic acid content of the fractionated, samples was observed until 12.1 mol mol^–1 of EPO, there was no further increase in their activity over 12.4 mol mol^–1 of EPO. On the other hand, an inverse relationship between thein vitro bioactivity and sialic acid content of EPO was observed. Also, we showed that thein vivo bioactivity of some fractions with low sialic acid contents was increased after treatment with 2,6-sialyltransferase, but thein vivo bioactivity of the other fractions with high sialic acid contents was either decreased or not affected.Abbreviations EPO erythropoietin - rHuEPO recombinant human erythropoietin - hCG human chorionic gonadotropin - BHK baby hamster kidney - CHO Chinese hamster ovary - NeuAc N-acetyl neuraminic acid - Gal galactose - HRCs hemolyser-resistant cells - WST-1 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Na - IEF isoelectric focusing - pI isoelectric point