A Mammalian Genetic Complementation Assay for Assessing Cellular Resistance to Genotoxic Compounds.

A complementation assay was developed to determine whether alleles of DNA repair genes are necessary for repairing specific types of damage. The assay was established by measuring the resistance capacity of Rad51d-deficient mouse embryonic fibroblasts (MEFs) transfected with mammalian expression constructs. Here, we describe the methods used to assess colony survival following the treatment of transfected cells with genotoxic compounds. This approach provides a time-efficient and stringent strategy to screen genetic alleles for identifying regions or specific amino acid residues critical for function or regulation of DNA repair pathways.

Agrobacterium-mediated transformation of Fusarium proliferatum.

Fusarium proliferatum is an important pathogen that is associated with plant diseases and primarily affects aerial plant parts by producing different mycotoxins, which are toxic to humans and animals. Within the last decade, this fungus has also been described as one of the causes of red root rot or sudden death syndrome in soybean, which causes extensive damage to this crop. This study describes the Agrobacterium tumefaciens-mediated transformation of F. proliferatum as a tool for the disruption of pathogenicity genes. The genetic transformation was performed using two binary vectors (pCAMDsRed and pFAT-GFP) containing the hph (hygromycin B resistance) gene as a selection marker and red and green fluorescence, respectively. The presence of acetosyringone and the use of filter paper or nitrocellulose membrane were evaluated for their effect on the transformation efficiency. A mean processing rate of 94% was obtained with 96 h of co-cultivation only in the presence of acetosyringone and the use of filter paper or nitrocellulose membrane did not affect the transformation process. Hygromycin B resistance and the presence of the hph gene were confirmed by PCR, and fluorescence due to the expression of GFP and DsRed protein was monitored in the transformants. A high rate of mitotic stability (95%) was observed. The efficiency of Agrobacterium-mediated transformation of F. proliferatum allows the technique to be used for random insertional mutagenesis studies and to analyze fungal genes involved in the infection process.

Hygromycin B-induced cell death is partly mediated by reactive oxygen species in rice (Oryza sativa L.).

The aminoglycoside antibiotic hygromycin B (Hyg) inhibits prokaryotic, chloroplast and mitochondrial protein synthesis. Because of the toxic effect of Hyg on plant cells, the HPT gene, encoding hygromycin phosphotransferase, has become one of the most widely used selectable markers in plant transformation. Yet the mechanism behind Hyg-induced cell lethality in plants is not clearly understood. In this study, we aimed to decipher this mechanism. With Hyg treatment, rice calli exhibited cell death, and rice seedlings showed severe growth defects, leaf chlorosis and leaf shrinkage. Rice seedlings also exhibited severe lipid peroxidation and protein carbonylation, for oxidative stress damage at the cellular level. The production of reactive oxygen species such as O2(·-), H2O2 and OH(·) was greatly induced in rice seedlings under Hyg stress, and pre-treatment with ascorbate increased resistance to Hyg-induced toxicity indicating the existence of oxidative stress. Overexpression of mitochondrial Alternative oxidase1a gene without HPT selection marker in rice enhanced tolerance to Hyg and attenuated the degradation of protein content, whereas the rice plastidial glutathione reductase 3 mutant showed increased sensitivity to Hyg. These results demonstrate that Hyg-induced cell lethality in rice is not only due to the inhibition of protein synthesis but also mediated by oxidative stress.

Plant genetic transformation efficiency of selected Malaysian rice based on selectable marker gene (hptII).

Rice is one of the most important cereal crops with great potential for biotechnology progress. In transformation method, antibiotic resistance genes are routinely used as powerful markers for selecting transformed cells from surrounding non-transformed cells. In this study, the toxicity level of hygromycin was optimized for two selected mutant rice lines, MR219 line 4 and line 9. The mature embryos were isolated and cultured on an MS medium with different hygromycin concentrations (0, 20, 40, 60, 80 and 100 mg L(-1)). Evidently, above 60 mg L(-1) was effective for callus formation and observed completely dead. Further there were tested for specific concentration (0-60). Although, 21.28% calli survived on the medium containing 45 mg L(-1) hygromycin, it seemed suitable for the identification of putative transformants. These findings indicated that a system for rice transformation in a relatively high frequency and the transgenes are stably expressed in the transgenic plants. Green shoots were regenerated from the explant under hygromycin stress. RT-PCR using hptII and gus sequence specific primer and Southern blot analysis were used to confirm the presence of the transgene and to determine the transformation efficiency for their stable integration in regenerated plants. This study demonstrated that the hygromycin resistance can be used as an effective marker for rice transformation.

Cre recombinase-mediated site-specific modification of a cellular genome using an integrase-defective retroviral vector.

Retroviral integrase is an enzyme responsible for the integration of retroviruses. A single mutation in the integrase core domain can severely compromise its integration ability, leading to the accumulation of circular retroviral cDNA in the nuclei of infected cells. We therefore attempted to use those cDNA as substrates for Cre recombinase to perform a recombinase-mediated cassette exchange (RMCE), thereby targeting retroviral vectors to a predetermined site. An expression unit containing a promoter, an ATG codon and marker genes (hygromycin resistance gene and red fluorescent protein gene) flanked by wild-type and mutant loxP sites was first introduced into cellular chromosome to build founder cell lines. We then constructed another plasmid for the production of integrase-defective retroviral vectors (IDRV), which contains an ATG-deficient neomycin resistance gene and green fluorescent protein gene, flanked by a compatible pair of loxPs. After providing founder cells with Cre and infecting with IDRV later, effective RMCE occurred, resulting in the appearance of G418-resistant colonies and a change in the color of fluorescence from red to green. Southern blot and PCR analyses on selected clones further confirmed site-specific recombination. The successful substitution of the original viral integration machinery with a non-viral mechanism could expand the application of retroviral vectors.

Disruption of gap junctions attenuates aminoglycoside-elicited renal tubular cell injury.

BACKGROUND AND PURPOSE: Gap junctions play important roles in the regulation of cell phenotype and in determining cell survival after various insults. Here, we investigated the role of gap junctions in aminoglycoside-induced injury to renal tubular cells. EXPERIMENTAL APPROACH: Two tubular epithelial cell lines NRK-E52 and LLC-PK1 were compared for gap junction protein expression and function by immunofluorescent staining, Western blot and dye transfer assay. Cell viability after exposure to aminoglycosides was evaluated by WST assay. Gap junctions were modulated by transfection of the gap junction protein, connexin 43 (Cx43), use of Cx43 siRNA and gap junction inhibitors. KEY RESULTS: NRK-E52 cells expressed abundant Cx43 and were functionally coupled by gap junctional intercellular communication (GJIC). Exposure of NRK-E52 cells to aminoglycosides, G418 and hygromycin, increased Cx43 phosphorylation and GJIC. The aminoglycosides also decreased cell viability that was prevented by gap junction inhibitors and Cx43 siRNA. LLC-PK1 cells were gap junction-deficient and resistant to aminoglycoside-induced cytotoxicity. Over-expression of a wild-type Cx43 converted LLC-PK1 cells to a drug-sensitive phenotype. The gap junction inhibitor alpha-glycyrrhetinic acid (alpha-GA) activated Akt in NRK-E52 cells. Inhibition of the Akt pathway enhanced cell toxicity to G418 and abolished the protective effects of alpha-GA. In addition, gentamycin-elicited cytotoxicity in NRK-E52 cells was also significantly attenuated by alpha-GA. CONCLUSION AND IMPLICATIONS: Gap junctions contributed to the cytotoxic effects of aminoglycosides. Modulation of gap junctions could be a promising approach for prevention and treatment of aminoglycoside-induced renal tubular cell injury.

A role for the non-phosphorylated form of yeast Snf1: tolerance to toxic cations and activation of potassium transport.

The Snf1/AMP-activated protein kinases play a key role in stress responses of eukaryotic cells. In the yeast Saccharomyces cerevisiae Snf1 is regulated by glucose depletion, which triggers its phosphorylation at Thr210 and concomitant increase in activity. Activated yeast Snf1 is required for the metabolic changes allowing starvation tolerance and utilization of alternative carbon sources. We now report a function for the non-activated form of Snf1: the regulation of the Trk high-affinity potassium transporter, encoded by the TRK1 and TRK2 genes. A snf1Delta strain is hypersensitive in high-glucose medium to different toxic cations, suggesting a hyperpolarization of the plasma membrane driving increased cation uptake. This phenotype is suppressed by the TRK1 and HAL5 genes in high-copy number consistent with a defect in K(+) uptake mediated by the Trk system. Accordingly, Rb(+) uptake and intracellular K(+) measurements indicate that snf1Delta is unable to fully activate K(+) import. Genetic analysis suggests that the weak kinase activity of the non-phosphorylated form of Snf1 activates Trk in glucose-metabolizing yeast cells. The effect of Snf1 on Trk is probably indirect and could be mediated by the Sip4 transcriptional activator.

Activation of delta-isoform of protein kinase C is required for oxidant-induced disruption of both the microtubule cytoskeleton and permeability barrier of intestinal epithelia.

Using monolayers of intestinal (Caco-2) cells, we showed that oxidants disassemble the microtubule cytoskeleton and disrupt barrier integrity (permeability) (Banan et al., 2000a). Because exposure of our parental cells to oxidants causes protein kinase C (PKC)-delta to be translocated to particulate fractions, we hypothesized that PKC-delta activation is required for these oxidant effects. Monolayers of parental Caco-2 cells were incubated with oxidant (H(2)O(2)) +/- modulators. Other cells were transfected with an inducible plasmid to stably overexpress PKC-delta or with a dominant negative plasmid to stably inhibit the activity of native PKC-delta. In parental cells, oxidants caused translocation of PKC-delta to the particulate (membrane + cytoskeletal) fractions, activation of PKC-delta isoform, increases in monomeric (S1) tubulin and decreases in polymerized (S2) tubulin, disruption of the microtubule cytoarchitecture, and loss of barrier integrity (hyperpermeability). In transfected cells, induction of PKC-delta overexpression by itself (3.5-fold over its basal level) led to oxidant-like disruptive effects. Disruption induced by PKC-delta overexpression was potentiated by oxidants. Overexpressed PKC-delta resided in particulate fractions, indicating its activation. Stable inhibition of native PKC-delta activity (98%) by dominant negative transfection substantially protected against all measures of oxidative disruption. We conclude that 1) oxidants induce loss of intestinal epithelial barrier integrity by disassembling the microtubules in large part through the activation of the PKC-delta isoform; and 2) overexpression and activation of PKC-delta is by itself a sufficient condition for disruption of these cytoskeleton and permeation pathways. Thus, PKC-delta activation may play a key role in intestinal dysfunction in oxidant-induced diseases such as inflammatory bowel disease.

Expression of hygR in transgenic mice causes resistance to toxic effects of hygromycin B in vivo.

Aminoglycoside antibiotics are indispensable for treatment of serious bacterial infections, and despite careful attention to dosage regimens, nephrotoxicity and ototoxicity still cause concern. In the present study, we tested whether side effects of aminoglycoside therapy could be limited by expression of prokaryotic genes of antibiotic resistance in vivo. We characterized the acute and tissue-specific toxicity of hygromycin B in transgenic mice bearing the hygromycin B phosphotransferase (hygR) gene under control of a constitutive promoter. We characterized the tissue-specific expression of hygR mRNA and also investigated the acute toxicity of hygromycin B in hygR and wild-type mice. The hygR mRNA reached its highest levels in brain and reached intermediate levels in spleen, muscle, kidney, liver and testis. The lowest levels were detected in heart and lungs. The hygR expression in transgenic animals caused an 89-fold increase in the approximate lethal dose of hygromycin B compared with wild-type mice. Serum biochemical analysis of hygR and wild-type mice treated with lethal doses of hygromycin B indicated liver and kidney damage measured as ALT, AST and BUN. On the morphological level, these changes led to acute tubular nephrosis in wild-type mice and acute liver damage in hygR mice. Our results show that constitutive expression of the bacterial hygR gene in transgenic mice in vivo confers resistance to hygromycin B.

Mutants of Neurospora crassa that alter gene expression and conidia development.

Several genes have been identified that are highly expressed during conidiation. Inactivation of these genes has no observable phenotypic effect. Transcripts of two such genes, con-6 and con-10, are normally absent from vegetative mycelia. To identify regulatory genes that affect con-6 and/or con-10 expression, strains were prepared in which the regulatory regions for these genes were fused to a gene conferring hygromycin resistance. Mutants were then selected that were resistant to the drug during mycelial growth. Mutations in several of the isolates had trans effects; they activated transcription of the corresponding intact gene and, in most isolates, one or more of the other con genes. Most interestingly, resistant mutants were obtained that were defective at different stages of conidiation. One mutant conidiated under conditions that do not permit conidiation in wild type.

Elevation of sister chromatid exchange frequency in transformed human fibroblasts following exposure to widely used aminoglycosides.

Aminoglycosides are a class of antibiotics that interfere with protein translation. Geneticin and hygromycin are two such agents, which have been shown to exhibit highly toxic effects in mammalian cells. Cloned bacterial genes, which inactivate these antibiotics, have facilitated the establishment of dominant selection systems, which are widely used in eukaryotic molecular genetics. We have examined the effect of aminoglycosides on the sister chromatid exchange (SCE) frequency in transformed human fibroblast cell lines. Geneticin and hygromycin were both found to increase SCE frequency in all cell lines examined, including a cell line derived from a patient with Bloom syndrome, a disorder exhibiting an elevated spontaneous SCE frequency. Induction was seen to occur in a dose-responsive manner and was also observed in cells expressing the resistance genes that inactivate the cellular toxicity of these antibiotics. The implications of these findings for somatic cell genetics and for human gene therapy protocols are discussed.

Hygromycin B therapy of a murine coronaviral hepatitis.

Hepatitis caused by mouse hepatitis virus (MHV-A59), a murine coronavirus, is accompanied by direct infection and replication of virus within the liver. We demonstrate here that the aminoglycoside hygromycin B is able to eliminate MHV-A59 infection from mouse peritoneal macrophages and cultured liver cells in vitro and is also able to reduce levels of virus replication and necrotic liver foci in vivo.

Construction of a fusion gene that confers resistance against hygromycin B to mammalian cells in culture.

Mouse L fibroblasts and other mammalian cells are killed by the translation inhibitor hygromycin B. We have modified the gene conferring resistance against hygromycin B in E. coli in such a way that it can be transcribed in mammalian cells from the promoter of the HSVtk gene. The resulting plasmid, pHMR272, was transfected into mouse L fibroblasts and HeLa cells by the calcium phosphate method and upon selection produced clones resistant against hygromycin B. The transfection rate was similar to that obtained with other selective markers. This plasmid is a useful addition to the relatively small number of dominant selectable markers available for mammalian cells.

[Effect of phytobacteriomycin, trichotecin, hygromycin B and levoristatin on some rat liver enzymes]. / Vliianie fitobakteriomitsina, trikhotetsina, gigromitsina B i levoristatina na nekotorye fermenty pecheni krys

In experiments with albino rats it was found that after administration of phytobacteriomycin, trichotecin, hygromycin B or levoristatin into the stomach in doses of 1/20 of LD50 activity of the microsomal enzymes of the liver cells significantly changed and the changes persisted within at least 2 weeks. The above antibiotics induced similar changes in the lysosome enzyme, i.e. acid phosphatase, providing an increase in its activity. Changes in the activity of succinate dehydrogenase (mytochondria indicator enzyme), glucose-6-phosphatase (ribosome indicator enzyme) and aspartate aminotransferase (cytoplasm indicator enzyme) were different for each antibiotic. It is concluded that the above antibiotics were capable of impairing on intoxication the enzymatic function of various cell microstructures, though the levels of the change direction may be different.