Identifying sites of simultaneous DNA replication in eukaryotes by N-methyl-N''-nitro-N-nitrosoguanidine multiple mutagenesis.

Summary N-methyl-N-nitro-N-nitrosoguanidine (NG) induces certain classes of multiple mutations in yeast at high frequency. By selecting for mutation at one locus (his4 or leu1) one frequently obtains double mutants where another mutation to temperature sensitivity has also been induced. This multiple mutagenesis exhibits a considerable specificity: for mutation at one particular locus there is a high chance that another mutation will be found in the same cell at one of a restricted number of other loci. For any given locus (e.g. his4) there is a spectrum of sites at which temperature-sensitivity mutations are coinduced. This spectrum differs for different loci, such that the spectrum of sites co-mutating with leul differs completely from that for sites co-mutating with his4. This NG-induced co-mutation is interpreted in terms of NG acting to enhance mutagenesis at sites of simultaneous DNA replication within the cell. The results so obtained indicate a very strict control over the order and timing of gene replication in Saccharomyces cerevisiae, and it is suggested that it is now possible to use NG double mutagenesis to try and locate origins of replication in yeast.     

Developmental regulation of processing alpha-mannosidases and "intersecting" N-acetylglucosaminyltransferase in Dictyostelium discoideum.

We have identified three developmentally regulated oligosaccharide-processing enzyme activities in Dictyostelium discoideum. Two different alpha-mannosidase activities present at extremely low levels in vegetative cells are expressed during development. The first of these activities (MI) rises sharply from 6 to 12 h of development whereas the second activity (MII) rises sharply from 12 to 18 h of development. MI acts on Man9GlcNAc, which it can degrade to Man5GlcNAc but is inactive toward p-nitrophenyl-alpha-D-mannoside (pnpMan). MII acts on pnpMan but not Man9GlcNAc. These activities are distinct from each other and from lysosomal alpha-mannosidase activity as demonstrated by pH optima, substrate specificity, sensitivity to inhibitors and divalent cations, developmental profiles, and solubility. The characteristics of these developmentally regulated alpha-mannosidase activities are similar to those of Golgi alpha-mannosidases I and II from higher eucaryotes, and they appear to catalyze the in vivo formation of processed asparagine-linked oligosaccharides by developed cells. In addition, developed cells have very low levels of a soluble alpha-mannosidase activity, which is the predominant activity in vegetative cells. This soluble vegetative alpha-mannosidase activity has properties that are reminiscent of the endoplasmic reticulum alpha-mannosidase from rat liver. The intersecting N-acetylglucosaminyltransferase activity that we have described recently in vegetative cells of D. discoideum (Sharkey, D. J., and Kornfeld, R. (1989) J. Biol. Chem. 264, 10411-10419) has a developmental profile that is distinct from that of either of the alpha-mannosidase activities. It has maximum activity at 6 h of development and decreases sharply to its minimum level by 12 h of development. The changes that occur in the levels of these three processing enzymes with development correlate well with the different arrays of asparagine-linked oligosaccharides found in early and late stages of development (Sharkey, D. J., and Kornfeld, R. (1991) J. Biol. Chem. 266, 18485-18497).     

Technetium labeling of monoclonal antibodies with functionalized BATOs: 2. TcCl(DMG)3CPITC labeling of B72.3 and NP-4 whole antibodies and NP-4 F(ab')2.

BATO (boronic acid adduct of technetium dioximes) complexes, TcCl(dioxime)3BR, were prepared in which the boron substituent (R) was the protein-reactive 2-carboxy-4-phenyl isothiocyanate (CPITC). The 99Tc complexes, where the dioxime was either dimethylglyoxime (DMG) or cyclohexanedione dioxime (CDO), were prepared and characterized. The 99mTc complex TcCl(DMG)3CPITC was prepared from a freeze-dried kit and used to label B72.3 (anti-TAG.72) and NP-4 (anti-CEA) whole antibodies, and the NP-4 F(ab')2 fragment. SDS-PAGE electrophoresis indicated that the labeling reagent was strongly bound to antibody. The labeled antibodies displayed high binding to affinity columns and good tumor uptake in GW39 tumor-bearing mice.     

Separation and measurement of direct and indirect effects of light on stomata

Conductance for water vapor, assimilation of CO₂, and intercellular CO₂ concentration of leaves of five species were determined at various irradiances and ambient CO₂ concentrations. Conductance and assimilation were then plotted as functions of irradiance and intercellular CO₂ concentration. The slopes of these curves allowed us to estimate infinitesimal changes in conductance (and assimilation) that occurred when irradiance changed and intercellular CO₂ concentration was constant, and when CO₂ concentration changed and irradiance was constant. On leaves of Xanthium strumarium L., Gossypium hirsutum L., Phaseolus vulgaris L., and Perilla frutescens (L.), Britt., the stomatal response to light was determined to be mainly a direct response to light and to a small extent only a response to changes in intercellular CO₂ concentration. This was also true for stomata of Zea mays L., except at irradiances < 150 watts per square meter, when stomata responded primarily to the depletion of the intercellular spaces of CO₂ which in turn was caused by changes in the assimilation of CO₂.     

Asparagine metabolism-key to the nitrogen nutrition of developing legume seeds

Asparagine accounted for 50 to 70% of the nitrogen carried in translocatory channels serving fruit and seed of white lupin (Lupinus albus L.). Rates of supply of the amide always greatly exceeded its incorporation as such into protein. An asparaginase (l-asparagine amido hydrolase EC 3.5.1.1) was demonstrated in crude extracts of seeds. In vitro activity was up to 5 mumoles of aspartate formed per hour per gram fresh weight at the apparent Km(Asn) value of 10 mM, and this more than accounted for the estimated rates of asparagine utilization in vivo. Asparaginase activity per seed increased 10-fold in the period 5 to 7 weeks after anthesis, coinciding with early stages of storage protein synthesis in the cotyledons.Double labeled ((14)C (U), (15)N (amide)) asparagine was fed to fruiting shoots through the transpiration steram. Fruit phloem sap analysis indicated that virtually all of the label was translocated to seeds in the form of asparagine. In young seeds (15)N from asparagine breakdown was traced to the ammonia, glutamine, and alanine of endospermic fluid, the (14)C appearing mainly in nonamino compounds. In the cotyledon-filling stage the C and N of asparagine was contributed to a variety of amino acid residues of protein.     

Single-Cell Analysis Reveals Early Manifestation of Cancerous Phenotype in Pre-Malignant Esophageal Cells

Cellular heterogeneity plays a pivotal role in a variety of functional processes in vivo including carcinogenesis. However, our knowledge about cell-to-cell diversity and how differences in individual cells manifest in alterations at the population level remains very limited mainly due to the lack of appropriate tools enabling studies at the single-cell level. We present a study on changes in cellular heterogeneity in the context of pre-malignant progression in response to hypoxic stress. Utilizing pre-malignant progression of Barrett’s esophagus (BE) as a disease model system we studied molecular mechanisms underlying the progression from metaplastic to dysplastic (pre-cancerous) stage. We used newly developed methods enabling measurements of cell-to-cell differences in copy numbers of mitochondrial DNA, expression levels of a set of mitochondrial and nuclear genes involved in hypoxia response pathways, and mitochondrial membrane potential. In contrast to bulk cell studies reported earlier, our study shows significant differences between metaplastic and dysplastic BE cells in both average values and single-cell parameter distributions of mtDNA copy numbers, mitochondrial function, and mRNA expression levels of studied genes. Based on single-cell data analysis, we propose that mitochondria may be one of the key factors in pre-malignant progression in BE.     

A comparison of two novel alcohol dehydrogenase enzymes (ADH1 and ADH2) from the extreme halophile Haloferax volcanii

Haloarchaeal alcohol dehydrogenases are exciting biocatalysts with potential industrial applications. In this study, two alcohol dehydrogenase enzymes from the extremely halophilic archaeon Haloferax volcanii (HvADH1 and HvADH2) were homologously expressed and subsequently purified by immobilized metal-affinity chromatography. The proteins appeared to copurify with endogenous alcohol dehydrogenases, and a double Δadh2 Δadh1 gene deletion strain was constructed to prevent this occurrence. Purified HvADH1 and HvADH2 were compared in terms of stability and enzymatic activity over a range of pH values, salt concentrations, and temperatures. Both enzymes were haloalkaliphilic and thermoactive for the oxidative reaction and catalyzed the reductive reaction at a slightly acidic pH. While the NAD⁺-dependent HvADH1 showed a preference for short-chain alcohols and was inherently unstable, HvADH2 exhibited dual cofactor specificity, accepted a broad range of substrates, and, with respect to HvADH1, was remarkably stable. Furthermore, HvADH2 exhibited tolerance to organic solvents. HvADH2 therefore displays much greater potential as an industrially useful biocatalyst than HvADH1.