Fragile Mutant Research Articles

EFFECT OF TEMPERATURE AND SODIUM CHLORIDE CONCENTRATION ON GROWTH OF Staphylococcus aureus MUTANTS

تضمنت هذه الدراسة تشخيص عزلات المكورات العنقودية (النوع العادي والمطفرة). وشخصت كل العزلات على اساس تخميرها لسكر المانيتول و افرازها لكل من coagulase و الكاتلايز. وقد تم اختيار سلاله ( 24 ) للدراسة وعرضت لطفرة بواسطة NTG للعزلات الحساسة للحرارة (TOF). وقد تم عزل الخلايا المطفره TOF بنجاح من خلال تعريض معلق الخلايا إلى 200 ملغ / مل من NTG لمدة 75 دقيقة. نتائج التطفير TOF أظهرت أن هذه الخلايا المطفرة كانت مطابقة لسلالة الأم مظهريا. نتيجة التطفير جعل الليزوزيم قادرة على تحليل جدار الخلية TOF مماثل لـlysostaphin. وقد اظهر نمط الحساسية للمضادات الحيوية لجميع العزلات المقاومة ومتعددة لبعض العزلات تم الكشف عنها. أظهرت النتائج أن عزلتين مطفرة كانت مقاومة للميثيسيلين، بنزاثين, البنسلين, كلوكساسيلين الصوديوم ولينكوميسين وأيضا هذه العزلات كانت حساسة للأموكسيسيلين، Cefotaxine الصوديوم ، الصوديوم وسيفاليكسين وتتيتراسايكلين ومتوسطة في مقاومة كلينداميسين. وأظهرت تأثير درجات الحرارة المختلفة إلى أن نمو السلالة الأم (سلالة رقم 24) تزداد مع زيادة درجة الحرارة والسلالات الطافرة (3M ، 10M) ليست مستقرة في درجات الحرارة المختلفة ونموها منخفضة جدا في 40 م. وأظهرت ان تأثير تراكيز كلوريد الصوديوم المختلفة على النمو من سلالة الأم (سلالة رقم 24) والسلالتين الطافرة (3M ، 10M) تزيد مع زيادة تركيز كلوريد الصوديوم ولكن تنخفض في تركيز كلوريد الصوديوم = 1.5 مولاري.

In-Cell Protein Folding - PAPS Synthases

Here, we study the stabilities of different naturally fragile mutants of 3' phosphoadenosine 5' phosphosulfate (PAPS) synthases. The goal is to establish a relationship between the stability of the mutants in the cellular environment and their enzymatic activity. In vitro stability measurements have suggested that protein stability is a major factor for cellular PAPS availability [1]. We aim to specifically analyze different cellular factors for in-cell PAPSS stability. One crucial factor is the macromolecular crowding effect. The cell is filled up to a volume of 40 % with macromolecules. Often, artificial macromolecular crowding agents are used to mimic these conditions. We previously studied macromolecular crowding effects via a thermodynamic analysis of the thermal unfolding of ubiquitin [2]. We observed enthalpic stabilization and entropic destabilization forces for all tested crowders. Further, we tested how such artificial cosolutes reflect the physicochemical properties of the complex cellular environment. Therefore, we developed a FRET-based macromolecular crowding sensor to study the crowding effect in living cells [3]. We found that the in-cell crowding effect is distributed heterogeneously and can change significantly upon osmotic stress. In comparison, we now used Fast Relaxation Imaging [4] to study the stability of PAPSS within the cellular environment and compare the results to in vitro crowding studies.

Drugging the p53 pathway: understanding the route to clinical efficacy

The tumour suppressor p53 is the most frequently mutated gene in human cancer, with more than half of all human tumours carrying mutations in this particular gene. Intense efforts to develop drugs that could activate or restore the p53 pathway have now reached clinical trials. The first clinical results with inhibitors of MDM2, a negative regulator of p53, have shown efficacy but hint at on-target toxicities. Here, we describe the current state of the development of p53 pathway modulators and new pathway targets that have emerged. The challenge of targeting protein-protein interactions and a fragile mutant transcription factor has stimulated many exciting new approaches to drug discovery.

Identification and Gene Mapping of a Fragile and Leaf-tip Dead Mutant fld1 in Oryza sativa

Identification and Gene Mapping of a Fragile and Leaf-tip Dead Mutant <i>fld1</i> in <i>Oryza sativa</i>

Air-drying kinetics affect yeast membrane organization and survival

The plasma membrane (PM) is a key structure for the survival of cells during dehydration. In this study, we focused on the concomitant changes in survival and in the lateral organization of the PM in yeast strains during desiccation, a natural or technological environmental perturbation that involves transition from a liquid to a solid medium. To evaluate the role of the PM in survival during air-drying, a wild-type yeast strain and an osmotically fragile mutant (erg6Δ) were used. The lateral organization of the PM (microdomain distribution) was observed using a fluorescent marker related to a specific green fluorescent protein-labeled membrane protein (Sur7-GFP) after progressive or rapid desiccation. We also evaluated yeast behavior during a model dehydration experiment performed in liquid medium (osmotic stress). For both strains, we observed similar behavior after osmotic and desiccation stresses. In particular, the same lethal magnitude of dehydration and the same lethal kinetic effect were found for both dehydration methods. Thus, yeast survival after progressive air-drying was related to PM reorganization, suggesting the positive contribution of passive lateral rearrangements of the membrane components. This study also showed that the use of glycerol solutions is an efficient means to simulate air-drying desiccation.

Heat-Induced Antigen Retrieval Applied in Zebrafish: Whole-Mount In Situ Immunofluorescence Microscopy

Whole-mount immunofluorescence technique provides a way to reveal integrated expression patterns of biological molecules in individuals. Well-documented morphological preservation ability in biology makes aldehydes the fixative of choice. Cross-linking among biocomponents and aldehydes is the key for maintaining morphology but masks the biological molecules for immunodetection. This study performs an easily accessible method by applying heat-induced retrieval, which can rescue the antigenicity of the proteins and also enhance the labeling sensitivity of the fluorescence dye in overfixed zebrafish embryos. The results show that the immunoreactivities of antibodies to myosin in the muscles, green fluorescent protein in the blood vessels and the nuclei in the cells can be recovered significantly, and the morphology of the zebrafish embryos, even the fragile mutants, is at the same time well maintained. Therefore, we provide a choice for antigen retrieval, which is effective for whole-mount immunofluorescence microscopy.

Transfection of multiple pulmonary cell types following intravenous injection of PEI-DNA in normal and CFTR mutant mice

The polycationic vector polyethylenimine (PEI) has been shown to be a powerful agent for transfecting the mouse lung after injection of plasmid-based polyplexes through the tail vein. These findings raise therapeutic prospects for a number of lung conditions. For such potentials to be realised, the precise identity of the transfected cells remains to be determined; however, so far, no ultrastructural analysis has been performed on PEI-transfected lungs. The definition of which pulmonary cells are transfected is particularly critical for certain pulmonary diseases which might require transfection of defined cell types such as epithelial cells for cystic fibrosis (CF). Here, we use a combination of light and electron microscopy to determine which cells are transfected in the lung after PEI-mediated gene delivery through the intravenous route. Furthermore, we extend the same experimental setting to a mouse model of CF to provide proof of principle that this approach can be used in genetic models of the disease. We show that within 18-20 h after injection through the tail vein, DNA/PEI complexes have already crossed the capillary barrier resulting in high levels of expression of reporter genes in the lungs. Transgene expression is observed in endothelial cells, in type I and type II pneumocytes, and in septal cells. Coexpression of the transgene and of the endogenous CF transmembrane conductance regulator (CFTR) gene is observed in some of the targeted epithelial cells. Levels and sites of expression are similar in normal and in CFTR-mutant mice. The results demonstrate that PEI-mediated gene delivery leads to transfection of epithelial cells beyond the endothelial barrier and show that this method can be used for lung gene delivery in CF fragile mutant mice.

Isolation and genetic characterization of a fragile plant mutant in rice (itOryza sauva) L.

The fragile rice mutant was isolated from an M2 population of indica variety Shuang Ke Zao (SKZ) treated with γ-rays, and designated asfpl (fragile plant 1) because of its fragile leaves and culms. To mapFP1 locus, an F2 mapping population was derived from a cross between thefpl and C-bao, a polymorphic japonic variety. The primary mapping result places theFP1 locus in an interval between two molecular markers, microsatellite marker RM16 (3.1 cM proximal toFP1) and STS marker G144a (9.1 cM distal toFP1) in the centromere region of chromosome 3. A CAPS marker C524a was further developed between RM16 and G144a, with 0.4 cM genetic distances to theFP1 locus, providing a practical starting point for constructing a BAC contig spanning theFP1 locus and cloning thefpl gene. Allelism test demonstratedthat fpl is allelic tobcl, a fragile rice mutant reported previously.

Non-wasteful fractionation of fragile yeast cells for the production of nutritional protein and other by-products

Saccharomyces cerevisiae 211 is a fragile yeast mutant whose cells grow only in media supplemented with osmotic stabilizer (1.6% NaCl), but which lyse spontaneously in water. This property provides a non-conventional way for isolation of nutritional protein and other products. We describe here a procedure based on the lysis ability of fragile yeasts for processing the biomass into several fractions. Cell lysis and downstream fractionation of the lysate do not include chemical or temperature treatment steps. The obtained protein fractions account for half of the starting biomass and contain 86% fully digestible protein and only 2% nucleic acids. The glycan fraction (with 83% polysaccharides) and the low molecular mass fraction are by-products of the procedure. The latter can be used as a nutritional media supplement in microbiology and as a source for purification of 5′-GMP, a potent flavour enhancer. The high rate of quantitative recovery and the mild conditions used to fractionate the biomass indicate the advantages of the fragile yeasts for production of nutritional protein and other products on a large scale by an efficient and non-wasteful technology.

Cell extensions in pkc1 mutants of Saccharomyces cerevisiae

To obtain information on cell wall synthesis and its relationship to morphology, we examined the induction of cell extensions of yeast upon the addition of isoamyl alcohol in osmotically fragile mutants that had mutations in genes related to the cell integrity pathway through activation of the mitogen-activated protein kinase cascade. We found that isoamyl alcohol induces cell extensions in pkc1 deletion mutants but not in mutants with mutations in genes positioned downstream or upstream of the PKC1 gene. These results suggest that Pkc1p functions not only in the integrity pathway but also in the induction. We characterized the elongated cells; many had two or more nuclei. We found no difference in cell surface structure between round and elongated cells from the results of chitin staining and cell wall extraction. Actin cytoskeleton was organized in elongated cells, as well as round cells. Cytochalasin D (0.08 mg/mL) inhibited the formation of actin cable but did not affect the induction of cell extensions.

Isolation and characterization of Schizosaccharomyces pombe fragile mutants.

Three Schizosaccharomyces pombe fragile mutants requiring the presence of an osmotic stabilizer to grow, that lyse when transferred into hypotonic solutions and that secrete to the extracellular medium more protein than the parental strain were isolated. In the three mutants, the fragile phenotype segregated in a Mendelian fashion, indicating a single chromosomal gene mutation, and behaved as a recessive character. By complementation analysis, the three fragile mutants fell in a single complementation group, defining the same gene (SRB1). Mutations of this gene are responsible for alterations in the cells such as fragile character, increase in the cell wall porosity, changes in the cell morphology and floc-forming ability. The study of the three srb1 alleles indicated that the degree of these alterations is proportional to a significant decrease in the galactomannan fraction of the mutants cell wall. The data presented in this report suggest that the product of the SRB1 gene is critical for the maintenance of the integrity and structure of Sz. pombe cell wall.

Schizosaccharomyces pombe fragile mutants as a host for heterologous protein production

In order to gain information about the potential interest of the Schizosaccharomyces pombe srb1 fragile mutants as a host for heterologous protein production, the extracellular secretion of homologous and heterologous invertases was investigated. Under catabolic derepression the fragile srb1 mutants released into the extracellular medium 5–6-fold more invertase than the parental strain. When transformed with the SUC2 gene, which codes for Saccharomyces cerevisiae invertase, the srb1-3 fragile mutant, grown under catabolic repression, released into the medium 3-fold more invertase than the wild-type transformant, even though the majority of the enzyme remained associated with the cell wall. Electrophoretic analysis revealed the presence in the fragile strains of some invertase forms with molecular weights smaller than their parallel wild-type strains, suggesting that the srb1 mutants may underglycosylate not only their homologous but also the heterologous proteins.

Isolation and characterization of Schizosaccharomyces pombe fragile mutants

Three Schizosaccharomyces pombe fragile mutants requiring the presence of an osmotic stabilizer to grow, that lyse when transferred into hypotonic solutions and that secrete to the extracellular medium more protein than the parental strain were isolated. In the three mutants, the fragile phenotype segregated in a Mendelian fashion, indicating a single chromosomal gene mutation, and behaved as a recessive character. By complementation analysis, the three fragile mutants fell in a single complementation group, defining the same gene (SRB1). Mutations of this gene are responsible for alterations in the cells such as fragile character, increase in the cell wall porosity, changes in the cell morphology and floc-forming ability. The study of the three srb1 alleles indicated that the degree of these alterations is proportional to a significant decrease in the galactomannan fraction of the mutants cell wall. The data presented in this report suggest that the product of the SRB1 gene is critical for the maintenance of the integrity and structure of Sz. pombe cell wall.

Isolation and Characterization of Schizosaccharomyces pombe Fragile Mutants

Three Schizosaccharomyces pombe fragile mutants requiring the presence of an osmotic stabilizer to grow, that lyse when transferred into hypotonic solutions and that secrete to the extracellular medium more protein than the parental strain were isolated. In the three mutants, the fragile phenotype segregated in a Mendelian fashion, indicating a single chromosomal gene mutation, and behaved as a recessive character. By complementation analysis, the three fragile mutants fell in a single complementation group, defining the same gene (SRB1). Mutations of this gene are responsible for alterations in the cells such as fragile character, increase in the cell wall porosity, changes in the cell morphology and floc-forming ability. The study of the three srb1 alleles indicated that the degree of these alterations is proportional to a significant decrease in the galactomannan fraction of the mutants cell wall. The data presented in this report suggest that the product of the SRB1 gene is critical for the maintenance of the integrity and structure of Sz. pombe cell wall.

Secretion of oligomeric Val8-human calcitonin by Saccharomyces cerevisiae

Monomeric human calcitonin (hCT) gene and oligomeric hCT genes composed of two, three or four head-to-tail linked monomers were fused in-frame to the yeast alpha-factor leader coding sequence wild-type and fragile mutant Saccharomyces cerevisiae strains were transformed with the constructed plasmids and the yield of recombinant protein secreted into the culture medium was measured. The yeast cells secreted equal (molar) amounts of all of the hCT variants. The recombinant proteins remained stable in the growth medium for at least 3 days. The fragile cells secreted about 30% more hCT as compared to the wild-type yeast cells.

Secretion of oligomeric Val8-human calcitonin bySaccharomyces cerevisiae

Monomeric human calcitonin (hCT) gene and oligomeric hCT genes composed of two, three or four head-to-tail linked monomers were fused in-frame to the yeast alpha-factor leader coding sequence wild-type and fragile mutant Saccharomyces cerevisiae strains were transformed with the constructed plasmids and the yield of recombinant protein secreted into the culture medium was measured. The yeast cells secreted equal (molar) amounts of all of the hCT variants. The recombinant proteins remained stable in the growth medium for at least 3 days. The fragile cells secreted about 30% more hCT as compared to the wild-type yeast cells.

Cell fusion of Saccharomyces cerevisiae fragile mutants

Fragile mutants of Saccharomyces cerevisiae are defective in the structure of the cell wall and plasma membrane. The mutant cells lyse in hypotonic solutions but grow exponentially when osmotic stabilizer is included in the medium. These mutants display a general increase in the permeability of the plasma membrane. We show here that fragile yeast cells of the same mating type can fuse without protoplast formation. The frequency of cell x cell fusion is lower than that observed for protoplast x protoplast fusion and can be significantly increased if the cells of one partner are converted to protoplasts. Microscopic observations and genetic analysis demonstrate that the hybrids obtained are fusion products. The fusion between fragile cells is explained in terms of the existence of local defects on their surface where the cell wall is thinner (or even missing), thus allowing a direct contact of cells by means of their plasma membranes.

Membrane transport in an osmotically fragile mutant of Saccharomyces cerevisiae.

Transport properties of the osmotically fragile strain VY1160 of Saccharomyces cerevisiae were compared with those of the parent S288c strain. Mediated diffusion of 6-deoxy-D-glucose was practically unaffected; membrane-potential-dependent transport of D-glucosamine was very much depressed in the fragile strain. The H+-driven transport of L-lysine and L-proline, as well as that of the hitherto uninvestigated D-glucose-6-phosphate, were also very depressed. 2-Deoxy-D-glucose transport displayed slightly different kinetic parameters. Primary H+ extrusion by the plasma membrane H-ATPase was not diminished although the ATP-splitting activity was depressed by about 50%. The overall proton-motive force (pmf) of the fragile mutant at pH 5.5 was only 20 mV while in the parent strain it was 108 mV. In parallel with this, spontaneous acidification of the external medium (a CO2-associated event) was only about 2% of that in the parent strain. The defect in this, together with the inability to stimulate transport protein synthesis by glucose, may account for the generally poorer transport performance of the fragile mutant.

Effect of changes in the osmolarity of the growth medium on Vibrio cholerae cells.

The rate and extent of lysis of Vibrio cholerae cells under nongrowing conditions were dependent on the osmolarity of the growth medium. Gross alterations in cellular morphology were observed when V. cholerae cells were grown in media of high and low osmolarity. The rate of lysis of V. cholerae cells under nongrowing conditions increased after treatment with chloramphenicol. Chloramphenicol-treated V. cholerae 569B cells showed formation of sphaeroplast-like bodies in medium of high osmolarity, but not in low osmolarity. Changes in the osmolarity of the growth medium also regulated the expression of the outer membrane proteins. This regulation was abolished if V. cholerae cells were grown in Pi-depleted medium. Analysis of the lytic behavior and composition of outer membrane proteins of an osmotically fragile mutant strain revealed a similar dependence on the osmolarity of the growth medium.

Uptake of DNA by fragile mutants of Saccharomyces cerevisiae

When Saccharomyces cerevisiae SY15 rho° mutant cells grown in media stabilized with 10% sorbitol were suspended in 2% sorbitol solutions, 60-70% of the population did not lyse and became permeable to native high molecular weight DNA. Maximal incorporation of DNA to DNase resistant state was measured after 60 min of incubation in presence of 5 μg/ml DNA and 10 mM CaCl2. These results suggest that the fragile mutants might be tested as hosts for transformation of whole yeast cells.