Functional genomics of the yeast DNA-damage response

High-throughput approaches are beginning to have an impact on many areas of yeast biology. Two recent studies, using different experimental platforms, provide insight into new pathways involved in the response of yeast to DNA damage.     

Cancer-associated genes can affect somatic intrachromosomal recombination early in carcinogenesis

The pKZ1 recombination mutagenesis model has provided a sensitive assay where we study somatic intrachromosomal recombination (SICR) as a mutation end-point. SICR is associated with non-homologous end-joining repair of double-strand breaks and can result in chromosomal inversions and deletions, both of which are common chromosomal aberrations identified in cancers. It has been difficult to study the effect of cancer-associated genes on chromosomal changes prior to tumour formation in vivo because of a lack of appropriate test systems. We hypothesised that cancer-associated genes play a role in formation of chromosomal aberrations and that the pKZ1 model would provide a system in which such a role could be studied in the initial steps of carcinogenesis. Transgenic tumour model mice were bred to pKZ1 mice to produce double transgenic animals. SICR inversion events were scored in mouse tissues at an early time, prior to evident tumour formation, and compared with endogenous pKZ1 SICR levels. Over-expression of the c-myc proto-oncogene resulted in a significant 2.1-fold increase in SICR in spleen. Loss of Msh2 and expression of the SV40 T antigen resulted in a significantly reduced SICR frequency (0.3 of the endogenous frequency in pKZ1 mice) in spleen and prostate respectively. Therefore SICR was affected in the case of all three cancer-associated genes studied. We hypothesise that the increase and decrease in SICR in the presence of cancer-associated genes results from incorrect repairing of double-strand breaks. The data presented here suggest that the pKZ1 model may provide a powerful tool for studying the effect of cancer-associated genes on chromosomal changes in the early stages of carcinogenesis.     

The tomato Cf-9 disease resistance gene functions in tobacco and potato to confer responsiveness to the fungal avirulence gene product avr 9

The Cf-9 gene encodes an extracytoplasmic leucine-rich repeat protein that confers resistance in tomato to races of the fungus Cladosporium fulvum that express the corresponding avirulence gene Avr 9. We investigated whether the genomic Cf-9 gene functions in potato and tobacco. Transgenic tobacco and potato plants carrying Cf-9 exhibit a rapid hypersensitive cell death response (HR) to Avr 9 peptide injection. Cf 9 tobacco plants were reciprocally crossed to Avr 9-producing tobacco. A developmentally regulated seedling lethal phenotype occurred in F1 progeny when Cf9 was used as the male parent and Avr 9 as the female parent. However, when Cf9 was inherited in the maternal tissue and a heterozygous Avr 9 plant was used as the pollen donor, a much earlier reaction was caused, leading to no germination of any F1 seed. Detailed analysis of the Avr 9-induced responses in Cf 9 tobacco leaves revealed that (1) most mesophyll cells died within 3 hr (compared with 12 to 16 hr in tomato); (2) the macroscopic HR was visible at an Avr 9 titer five times lower than that which caused visible symptoms in tomato; (3) the HR invariably extended into noninjected panels of the tobacco leaf; (4) no HR occurred in leaves of young tobacco plants; (5) in older plants, the HR was dramatically enhanced by sequential Avr 9 challenges; and (6) coexpression of a salicylate hydroxylase transgene (nahG) from Pseudomonas putida reduced the severity of the macroscopic leaf HR and also restored germination to Cf 9 x 35S:Avr 9 F1 seedlings. Simultaneous introduction of Cf-9 homologs (Hcr 9-9 genes A and B or D) along with the native Cf-9 gene did not alter the responses that were specifically induced by Avr 9. Various ways to use the Cf-9-Avr 9 gene combination to engineer broad-spectrum disease resistance in several solanaceous species are discussed.     

In Situ Bioremediation of Carbon Tetrachloride: Field Test Results

Results of a 7-month field test of in situ bioremediation of carbon tetrachloride (CT) under denitrifying conditions are reported. The demonstration was conducted in a portion of a several-square-mile CT and nitrate plume. Pretest CT and nitrate levels were 12.5 ± 0.14 μM and 3.87 ± 0.26 mM, respectively. During the test, the CT concentration dropped by 3.71 ± μM, representing an estimated total of 1.42 kg of CT destroyed. The total quantities of acetate and nitrate injected during the demonstration were 221 and 300 kg, respectively. Nitra injection was composed of short-duration, high-concentration pulses added with acetate pulses, and continuously injected nitrate that was present in the surrounding groundwater. Biomass was distributed successfully within the flow field without fouling the injection well. Levels of planktonic denitrifiers increased 10- and 5-fold in monitoring wells 3 and 6 m downstream from the injection well, respectively. A distributed growth pattern was indicated through reductions in the concentrations of acetate, nitrate, and nitrite between these wells. Chloroform (CF) production was controlled by adjusting acetate and nitrate pulsing to keep low levels of nitrate in most of the flow field. Under this regime only 1 mol% of transformed CT appeared as CF. In contrast, approximately 33 mol% of CT transformed to appear as CF when nutrient-feeding conditions were adjusted so that nitrate was consistently absent.     

Distribution of cell surface saccharides on pancreatic cells

We describe here a simple, general procedure for the purification of a variety of lectins, and for the preparation of lectin-ferritin conjugates of defined molar composition and binding properties to be used as probes for cell surface saccharides. The technique uses a “universal” affinity column for lectins and their conjugates, which consists of hog sulfated gastric mucin glycopeptides covalently coupled to agarose. The procedure involes: (a) purification of lectins by chromatography of aqueous extracts of seeds or other lectin-containing fluids over the affinity column, followed by desorption of the desired lectin with its hapten suge; (b) iodination of the lectin to serve as a marker during subsequent steps; (c) conjugation of lectin to ferritin with glutaraldehyde; (d) collection of active lectin-ferritin conjugates by affinity chromatography; and (e) separation of monomeric lectin-ferritin conjugates from larger aggregates and unconjugated lectin by gel chromatography. Based on radioactivity and absorbancy at 310 nm for lectin and ferritin, respectively, the conjugates consist of one to two molecules of lectin per ferrritin molecule. Binding studies of native lectins and their ferritin conjugates to dispersed pancreatic acinar cells showed that the conjugation procedure does not significantly alter either the affinity constant of the lectin for its receptor on the cell surface or the number of sites detected.     

Ethanol inhibition of antigen-induced histamine release from guinea pig lung: correlation with intracellular levels of cyclic nucleotides.

The effect of ethanol on histamine release from lungs of sensitized guinea pigs was studied in conjunction with measurements of tissue concentrations of cyclic AMP and cyclic GMP. Addition of antigen $\text{in vitro}$ elicited a rapid increase in cyclic AMP and cyclic GMP and stimulated release of histamine. Ethanol (2%) inhibited antigen-induced release of histamine over 95% and completely inhibited the increase in both cyclic nucleotides. The activity of cyclic AMP-dependent protein kinase was only slightly affected by ethanol.Metiamide blocked the ovalbumin stimulated increase in cyclic AMP but not cyclic GMP. Pyrilamine did not prevent the rise in either cyclic nucleotide. This suggests that the antigen-induced rise in cyclic AMP is an indirect result of histamine released from the tissue. The inability of H₁ and H₂ receptor antagonists to affect antigen-induced elevation of cyclic GMP in sensitized lung fragments suggests that an elevation in cyclic GMP might be either a primary event in the mediator release sequence or secondary to the release of a mediator other than histamine. The ability of ethanol to inhibit mediator release might be due to its capacity to attenuate the antigen-induced elevation of cyclic GMP in sensitized lung.     

The chiroptical properties of proteins. III. Adenylate kinase

The CD spectrum of the enzyme adenylate kinase has been investigated. Theoretical calculations, based on the x-ray crystal structure, have been carried out by means of an origin independent matrix formalism. The entire molecule was included in the calculations in the sense that essentially all electronic transitions that occur at wavelengths longer than 185 nm were included in the basis set. A linear dielectric function was utilized to evaluate the intertransition coupling potentials. The results of the theoretical calculations were in reasonable agreement with experimental CD spectra of the molecule.     

A protease-activatable luminescent biosensor and reporter cell line for authentic SARS-CoV-2 infection

Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.     

A platform for high-throughput molecular characterization of recombinant monoclonal antibodies

We describe quantitative characterization of a sample preparation platform for rapid and high-throughput analysis of recombinant monoclonal antibodies (MAbs) and their post-translational modifications. MAb capture, desalting and in situ reduction/alkylation were accomplished by sequential adsorption of analyte to solid phase beads (protein A, reverse-phase) suspended in microtiter plate wells. Following elution and rapid tryptic digestion in the presence of acid-labile surfactant (RapiGest), peptides were fractionated by stepwise elution from reverse-phase pipet tips and the fraction containing Fc N-glycopeptides isolated. Direct quantitative analysis of the relative abundance of peptide glycoforms by MALDI-TOF MS in linear mode closely correlated with normal phase HPLC analysis of fluorophore labeled N-glycans released by PNGaseF.