Mutational spectrum and recombinogenic effects induced by aminofluorene adducts in bacteriophage M13

Double-stranded replicative form (RFI) DNA of bacteriophage M13 strain M13mp10 which carries partial lacZ gene has been modified in vitro to various extents with N-hydroxy-2-amino-fluorene (N-OH-AF) and then transfected into E. coli cells. High-performance liquid chromatography (HPLC) analysis results demonstrate that the sole adduct (95%) formed in modified DNA is N-(deoxyguanosine-8-yl)-2-aminofluorene (dG-C8-AF). Approximately 20 adducts per RFI molecule constitute 1 lethal event when plaque-forming ability is assayed on E. coli cells which have received no prior SOS induction. The mutagenicity of dG-C8-AF adducts was assayed by measuring loss of beta-galactosidase activity as a function of adducts per molecule. A dose-dependent increase in Lac- mutants was observed, with a 4-fold increase in mutants per survivor at 30 adducts/molecule. The mutations produced, characterized by DNA sequencing, occur predominantly at either G or C positions different from those observed in the spontaneous mutant spectrum. Restriction-mapping results show that in our assay system, dG-C8-AF adducts induce a previously unreported recombinogenic activity.     

A map of photolytic and tryptic cleavage sites on the beta heavy chain of dynein ATPase from sea urchin sperm flagella

NH2-terminal analysis of the alpha and beta heavy chain polypeptides (Mr greater than 400,000) from the outer arm dynein of sea urchin sperm flagella, compared with that of the 230,000- and 200,000-Mr peptides formed upon photocleavage of dynein by irradiation at 365 nm in the presence of vanadate and ATP, shows that the NH2 termini of the intact chains are acetylated and that the 230,000- and 200,000 Mr peptides constitute the amino- and carboxy-terminal portions of the heavy chains, respectively. Tryptic digestion of the beta heavy chain is known to separate it into two particles, termed fragments A and B, that sediment at 12S and 6S (Ow, R. A., W.-J. Y. Tang, G. Mocz, and I. R. Gibbons, 1987. J. Biol. Chem. 262:3409-3414). Immunoblots against monoclonal antibodies specific for epitopes on the beta heavy chain, used in conjunction with photoaffinity labeling, show that the ATPase-containing fragment A is derived from the amino-terminal region of the beta chain, with the two photolytic sites thought to be associated with the purine-binding and the gamma-phosphate-binding areas of the ATP-binding site spanning an approximately 100,000 Mr region near the middle of the intact beta chain. Fragment B is derived from the complementary carboxy-terminal region of the beta chain.     

Epitopes of an influenza viral peptide recognized by antibody at single amino acid resolution

Antibodies raised against the synthetic peptide corresponding to the carboxy-terminal 24 amino acids (305-328) of the heavy chain of the hemagglutinin molecule of influenza virus A/X-31 (H3) bind this peptide at three antigenic sites. These sites were identified by assaying binding of polyclonal BALB/c mouse antipeptide sera to the complete set of all possible di-, tri, tetra-, penta-, hexa-, hepta-, and octapeptides homologous with the 24-residue sequence. Individual epitopes were defined and essential residues identified by testing the binding of monoclonal antibodies to sets of peptide analogues in which every one of the homologous residues was replaced in turn by each of the 19 alternative genetically coded amino acids. The immunodominant epitope was shown to be a linear sequence of five amino acids, 314LKLAT318. Replacement of any one of these residues with any other amino acid resulted in loss of antibody binding, indicating that all five are essential to the interaction and that they are probably contact residues. Another antigenic site contains at least two overlapping epitopes: polyclonal sera recognize predominantly an epitope or epitopes encompassed by the linear sequence 320MRNVPEKQT328, whereas the epitope defined by a particular monoclonal antibody comprises the seven amino acids 322NVPEKQT328, of which N322, E325, and Q327 were implicated as contact residues.     

水稻原生质体产生细胞团的冰冻保存和冻后再生植株形成

水稻(Oryza sativa L.)原生质体产生的细胞团加上10-20％的二甲亚枫(DMSO)和10-20％的蔗糖,置于液氮中保存。冻后细胞生存率达到对照的40-50％。存活的细胞在附加2×10~(-5)mol/l 2,4-D 的Linsmier-Skoog(Ls)固体培养基上再生长,然后将形成的愈伤组织块转到附加10~(-6)mol/l NAA,4×10~(-6)mol/l 激动素和10~(-6)mol/l 2 IP 及8％的蔗糖的 LS培养基上分化出芽并形成植株。     

Vimentin expression in oocytes, eggs and early embryos of Xenopus laevis

Immunocytochemical studies using a monoclonal anti-porcine vimentin antibody reveal a well-organized pattern of staining in Xenopus laevis oocytes, eggs and early embryos. The positions of Xenopus vimentin and desmin in two-dimensional (2D) polyacrylamide gels were first established by immunoblotting of muscle Triton extracts with anti-intermediate filament antibodies (anti-IFA), which cross-react with all intermediate filament proteins (IFPs). The anti-porcine vimentin reacts with vimentin and desmin in muscle 2D immunoblots, but only reacts with one polypeptide in oocyte blots in the position predicted for vimentin (Mr 55 x 10(3), pI 5.6). Using an anti-sense probe derived from a Xenopus vimentin genomic clone in RNase protection assays, we show that expression of vimentin begins in previtellogenic oocytes. The level of expression remains constant throughout oogenesis and in unfertilized eggs. These data suggest that vimentin is expressed in oocytes and eggs. Most interestingly, the immunocytochemical results also show that vimentin is present in the germ plasma of oocytes, eggs and early embryos. It is therefore possible that vimentin has an important role in the formation or behaviour of early germ line cells.     

Changes in lipid content and composition during the development of N-nitrosodiethylamine induced hepatocarcinoma

Alterations in lipid content and composition in the N-nitrosodiethylamine-induced hepatocarcinoma were investigated. Rats were administrated with N-nitrosodiethylamine in the drinking water for 12 weeks followed by normal tap water for another 6 weeks. The cholesterol content in the liver was increased shortly after the administration of N-nitrosodiethylamine and remained elevated after the removal of the nitrosoamine from the water. The phosphatidylethanolamine level was elevated during N-nitrosodiethylamine administration with a concomitant reduction in phosphatidylcholine level. Lysophosphatidylcholine and sphingomyelin levels were increased during the last four weeks of the study. The level of phosphatidylinositol was substantially reduced after eight weeks of N-nitrosodiethylamine treatment, and remained low during the post-treatment period. We postulate that changes in lysophosphatidylcholine and sphingomyelin may be a compensatory mechanism for maintaining the asymmetrical distribution of choline-containing lipids in the outer leaflet of the membrane. The elevated level of cholesterol may be a useful indicator for the early detection of N-nitrosodiethylamine-induced hepatocarcinoma.     

Generation of 19 STS markers that can be anchored at specific sites on human chromosome 21.

Sequence-tagged sites (STSs) are short stretches of DNA that can be specifically detected by the polymerase chain reaction (PCR) and can be used to construct long-range physical maps of chromosomal DNA. These STSs can be detected by PCR assays developed by reference to data obtained from the sequencing of restriction fragment length polymorphism-DNA markers for chromosome 21, which were derived from recombinant lamba-phage and plasmid clones made from DNA of a human-hamster hybrid cell line. In this report, we describe the generation of 19 new STSs that are specific for human chromosome 21.     

Several CD4 domains can play a role in human immunodeficiency virus infection in cells.

The human immunodefiency virus (HIV) uses the human CD4 glycoprotein as a receptor for infection of susceptible cells. Cells expressing a series of mutated forms of the CD4 gene have shown a variability in their ability to support replication of three HIV type 1 (HIV-1) and three HIV-2 strains. Moreover, when different stages of virus production were examined by a variety of assays, a consistent delay was observed in all cell lines containing CD4 mutants compared with those with intact full-length CD4. Cells expressing the CD4.415 mutant (modified at the serine 415 corresponding to a phosphorylation site of the cytoplasmic domain) showed only a minimal effect on virus replication. Cells expressing CD4.403 and CD4.401 mutants (lacking the whole cytoplasmic domain) manifested a moderate delay in production of virus progeny. The most substantial effect on HIV replication was observed in cells expressing a chimeric hybrid containing sequences corresponding to the first 177 residues of the N-terminal CD4 fused to CD8 sequences encoding the hinge, transmembrane, and cytoplasmic domains of the human CD8. Furthermore, in a cell-to-cell contact assay, fusion was absent when the CD4 proximal membrane domain was replaced by the CD8 counterpart. In addition, a strong correlation between the down-modulation of the surface CD4 and HIV expression was observed. These observations suggest that in addition to the known binding region, other domains of CD4 could play an important role in regulating HIV entry of cells.     

Biologic markers in ethylene oxide-exposed workers and controls

Ethylene oxide (EtO) is an alkylating agent and a model direct-acting mutagen and carcinogen. This study has evaluated a panel of biologic markers including EtO-hemoglobin adducts (EtO-Hb), sister-chromatid exchanges (SCEs), micronuclei, chromosomal aberrations (CAs), DNA single-strand breaks (SSB) and an index of DNA repair (ratio of UDS to NA-AAF-DNA binding) in the peripheral blood cells of 34 workers at a sterilization unit of a large university hospital and 23 controls working in the university library. Comprehensive environmental histories were obtained on each subject including detailed occupational and smoking histories. Industrial hygiene data obtained prior to the study and personal monitoring during the 8 years preceding the study showed that workers were subject to low-level exposure near or below the current Occupational Safety and Health Administration (OSHA) standard of 1 ppm (TWA). Personal monitoring data obtained during 2 weeks prior to blood sampling were uniformly less than 0.3 ppm (TWA). After adjusting for smoking, EtO workplace exposure was significantly (p less than 0.001) associated with EtO-Hb (a carcinogen-protein adduct) and 2 measures of SCEs [the average number of SCEs/cell (SCE50) and the number of high frequency cells (SCEHFC)]. There was an apparent suppression of DNA repair capacity in EtO-exposed individuals as measured by the DNA repair index; i.e., the ratio of unscheduled DNA synthesis (UDS) and NA-AAF-DNA binding (p less than 0.01). No association of DNA repair index with smoking was found. Another important finding of this study is the highly significant correlation between EtO-Hb adduct levels and SCEHFC (p less than 0.01) and SCEs (p less than 0.02) which provides evidence of a direct link between a marker of biologically effective dose and markers of genotoxic response. In contrast, micronuclei, CAs and SSBs were not significantly elevated in the workers. The activity of the u-isoenzyme of glutathione-S-transferase (GT) was measured as a possible genetic marker of susceptibility and a modulator of biomarker formation. However, possibly because of confounding by age, no significant relationships were found between GT and any of the exposure-related markers by ANOVA or among other independent variables by regression. This study demonstrates significant effects of low-level EtO exposure, independent of smoking history, near or below 1 ppm on multiple biomarkers and suggests that the current OSHA standard may not be adequately protective. Previously described effects of smoking on EtO-Hb adducts, SCEs and SCEHFC were also seen in this study.(ABSTRACT TRUNCATED AT 400 WORDS)