Comparisons of tests for aneuploidy

The fundamental problems that face us in the development of suitable assay systems for the detection of potentially aneugenic (aneuploidy-inducing) chemicals include: (a) the diversity of cellular targets and mechanisms where perturbations of structure and function may give rise to changes in chromosome number, and (b) the phylogenetic differences that exist between species in their mechanism and kinetics of cell division and their metabolic profiles. A diverse range of assay systems have been developed, which have been shown to have potential for use in the detection of either changes in chromosome number or of perturbations of the events which may be causal in the induction of aneuploidy.

Chromosome number changes may be detected cytologically by karyotypic analysis, or by the use of specialised strains in which aneuploid progeny may be observed due to phenotypic differences with aneuploid parental cells or whole organisms. Techniques for the detection of cellular target modifications range from in vitro studies of tubulin polymerisation to observations of the behaviour of various cellular organelles and their fidelity of action during the division cycle.

The diversity of mechanisms which may give rise to aneuploidy and the qualitative relevance of events observed in experimental organisms compared to man make it unlikely that the detection and risk assessment of the aneugenic activity of chemicals will be possible using a single assay system. Optimal screening and assessment procedures will thus be dependent upon the selection of an appropriate battery of predictive tests for the measurement of the potentially damaging effects of aneuploidy induction.     

Purification and characterization of glutathione reductase from corn mesophyll chloroplasts

Differences in the apparent molecular weights of the subunits of glutathione reductase (EC 1.6.4.2) from pea chloroplasts and corn mesophyll chloroplasts have been recently reported. In order to more fully describe the differences between the enzymes from these two sources, glutathione reductase from the mesophyll chloroplasts of corn seedlings ( Zea mays L. cv. G-4507) has been purified 200-fold by affinity chromatography using adenosine 2',5'-disphosphate agarose. The purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein)^-1 min^-1. The native enzyme had a relative molecular weight of 190 ± 30 kDa and exhibited polypeptides of 65, 63, 34, and 32 kDa when separated on sodium dodecylsulfate-polyacrylamide gels. Comparisons of the results from electroblotting, native molecular weight and subunit molecular weight analyses suggest that the enzyme exists as a heterotetramer. Optimal enzyme activity was obtained at pH 8 in N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES-NaOH) buffer. The sulfhydryl reagent, n -ethylmaleimide, inhibited enzymatic activity when incubated in the presence of NADPH while no inhibition was detected with oxidized glutathione in the incubation mixture. Reduced glutathione (5 m M ) inactivated the enzyme by 50%. This inactivation followed first order kinetics with a rate constant of 0.0028 s^-1. The enzyme was also inactivated by NADPH. The inactivation reached ca 90% within 30 min and followed first order kinetics with a rate constant of 0.0015 s^-1.     

Drought Stress and Elevated CO(2) Effects on Soybean Ribulose Bisphosphate Carboxylase Activity and Canopy Photosynthetic Rates

Soybean (Glycine max [L.] cv Bragg) was grown at 330 or 660 microliters CO₂ per liter in outdoor, controlled-environment chambers. When the plants were 50 days old, drought stress was imposed by gradually reducing irrigation each evening so that plants wilted earlier each succeeding day. On the ninth day, as the pots ran out of water CO₂ exchange rate (CER) decreased rapidly to near zero for the remainder of the day. Both CO₂-enrichment and drought stress reduced the total (HCO₃⁻/Mg²⁺-activated) extractable ribulose-1,5-bisphosphate carboxylase (RuBPCase) activity, as expressed on a chlorophyll basis. In addition, drought stress when canopy CER values and leaf water potentials were lowest, reduced the initial (nonactivated) RuBPCase activity by 50% compared to the corresponding unstressed treatments. This suggests that moderate to severe drought stress reduces the in vivo activation state of RuBPCase, as well as lowers the total activity. It is hypothesized that stromal acidification under drought stress causes the lowered initial RuBPCase activities. The K_m(CO₂) values of activated RuBPCase from stressed and unstressed plants were similar; 15.0 and 12.6 micromolar, respectively. RuBP levels were 10 to 30% lower in drought stressed as compared to unstressed treatments. However, RuBP levels increased from near zero at night to around 150 to 200 nanomoles per milligram chlorophyll during the day, even as water potentials and canopy CERs decreased. This suggests that the rapid decline in canopy CER cannot be attributed to drought stress induced limitations in the RuBP regeneration capability. Thus, in soybean leaves, a nonstomatal limitation of leaf photosynthesis under drought stress conditions appears due, in part, to a reduction of the in vivo activity of RuBPCase. Because initial RuBPCase activities were not reduced as much as canopy CER values, this enzymic effect does not explain entirely the response of soybean photosynthesis to drought stress.     

Characterization of the Erwinia carotovora pelB gene and its product pectate lyase.

The pelB gene encodes pectate lyase B, one of three pectate lyases identified in Erwinia carotovora EC. Pectate lyase B was purified from Escherichia coli containing the pelB gene on a recombinant plasmid. The activity of the protein was optimal at a pH of 8.3. The amino acid composition, N-terminal amino acid sequence, and C-terminal peptide sequence were determined and compared with the polypeptide sequence deduced from the DNA sequence of pelB. Purified pectate lyase B started at amino acid 23 of the predicted sequence, suggesting that a 22-amino-acid leader peptide had been removed. Pectate lyase B of E. carotovora EC and pectate lyase B of E. chrysanthemi EC16 contain 352 and 353 amino acids, respectively (N. T. Keen, S. Tanaki, W. Belser, D. Dahlbeck, and B. Staskawicz, J. Bacteriol. 168:595-606, 1986). The two proteins are 72% homologous on the basis of DNA sequence data, and 75% of the amino acids are identical.     

Characterization of three group A klebicin plasmids: localization of their E colicin immunity genes

We have investigated the immunity to E colicins conferred by three group A klebicin plasmids. pP5a, which encodes klebicin A1-P5, like pClo-DF13, confers immunity to colicin E6 on Escherichia coli K12, whilst pP5b and pP3, which encode klebicins A2-P5 and A3-P3 respectively, both confer immunity to colicin E3. We have determined the restriction endonuclease and functional maps of the three group A klebicin plasmids. By sub-cloning and transposon mutagenesis we have investigated the relationship between the klebicin immunity and the E colicin immunity conferred by these plasmids. The colicin E6 and the klebicin A1 immunity are encoded by a single gene present on pP5a. The colicin E3 and the klebicin A2 immunity are encoded by a single gene present on pP5b. The colicin E3 and the klebicin A3 immunity are encoded by separate genes present on pP3. Recombinant pML8412, which is derived from the ColE6-CT14 plasmid and encodes colicin E6 immunity, confers klebicin A1-P5 immunity upon Klebsiella pneumoniae UNF5023. Recombinant pKC23, which is derived from the ColE3-CA38 plasmid and confers colicin E3 immunity, confers immunity to klebicin A2-P5, but not to klebicin A3-P3.     

Characterization of the phospholipid and fatty acid composition of Sendai virus

The lipid composition of Sendai virus, propagated in chicken eggs, was analyzed by high performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and gas-liquid chromatography (GLC). Phosphatidylcholine was found to be the dominant phospholipid (37.3%) with phosphatidylethanolamine (26.8%) and phosphatidylserine (12.0%) also present in significant amounts. Analysis of the fatty acid methyl esters revealed that the dominant fatty acids in total phospholipid were: C16:0 (17.6%), C18:0 (15.4%), C18:1 (n-9) (22.0%), and C24:0 (6.0%). Cardiolipin, phosphatidylserine, and sphingomyelin contained higher levels of saturated fatty acids relative to phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine.