Isolation of high molecular weight DNA from wholeEuglena cells

Summary A method for isolating high quality DNA from wholeEuglena cells is described. The procedure consists in: the weakening of the cell pellicle in glycerol avoiding the mechanical disruption of cells and shearing damage in DNA molecules; the decondensation ofEuglena compact chromatin directly inside the cells; the complete dissociation of cells and nucleoproteins in sarkosyl detergent; the optional digestion of proteins and RNA with DNase-free enzymes and the final purification of DNA by isopycnic banding in CsCl gradients. Degradation of DNA is prevented all along the extraction procedure by glycerol, antioxydants, EDTA and sarkosyl detergent. Using the enzymatic digestion step, DNA containing few single-stranded nicks is obtained with a yield approaching 100%. DNA with no single-stranded nick could be obtained with a 35% yield when the enzymatic digestion step was omitted. In both cases, the double-stranded DNA has an average molecular weight equal or greater than 6×10⁷. It is free of contaminants and could be easily digested with restriction enzymes. After digestion with Eco RI and size-fractionation in agarose gel this DNA has permitted specific hybridization of the rDNA sequences with a radioactive rRNA probe.Abbreviations Kbp kilobasepairs - Kb kilobases     

Topological orientation of mitochondrial GDPmannose: dolichyl-monophosphate mannosyltransferase in the outer membrane

Previous studies have shown the existence of an autonomous mitochondrial GDPmannose: dolichylmonophosphate mannosyltransferase, located in mitochondiral outer membrane of liver cells. As nothing is known about glycosylation sites in mitochondria, we have investigated the topological orientation of this enzyme in intact mitochondria, using controlled proteolysis with trypsin. Mitochondria were purified sequentially by mild ultrasonic treatment and sucrose density gradient. Purity and homogeneity of mitochondrial fraction were assessed by electron microscopy and specific marker enzymes measures. Our data provide evidence for a mitochondrial GDPmannose: dolichylmonophosphate mannosyltransferase facing the cytoplasmic side of the outer membrane. However, the exposure of this enzyme to the water phase has been shown to be dependent on the ionic strength of the environment.     

Evolutionary divergence of enterobacterial genes coding for the glucose phosphotransferase system components

Abstract The DNA relatedness of 64 enterobacterial species to Escherichia coli genes pts I, pts H, crr , pts G, and pts LPM, was determined by quantitative filter hybridization. DNA relatedness was expressed relative to E. coli K-12 DNA. Enterobacterial DNAs were 0 to 100% related to E. coli genes and the level of relatedness (except for crr data) reflected the known taxonomic (phylogenetic) position of species with respect to E. coli . When pts I relatedness data were plotted against pts H data, correlation was excellent. In pts G versus pts LPM plots, the data points (species) were scattered along the diagonal with a large gap separating E. coli strains (80–100% relatedness to both probes) from the 63 other species (1 to 40% relatedness to E. coli genes). Serratia (9 species), Buttiauxella agrestis , and Klebsiella planticola gave higher relatedness values with crr probe than with the other probes tested.     

SIMS microscopy in the biomedical field.

We attempted to indicate the requirements for biomedical applications of SIMS microscopy. Sample preparation methodology should preserve both the structural and the chemical integrity of the tissue. Furthermore, it is often necessary to correlate ionic and light microscope images. This implies a common methodological approach to sample preparation for both microscopes. The use of low or high mass resolution depends on the elements studied and their concentrations. To improve the acquisition and processing of images, digital imaging systems have to be designed and require both ionic and optical image superimposition. However, the images do not accurately reflect element concentration; a relative quantitative approach is possible by measuring secondary ion beam intensity. Using an internal reference element (carbon) and standard curves the results are expressed in micrograms/mg of tissue. Despite their limited lateral resolution (0.5 microns) the actual SIMS microscopes are very suitable for the resolution of biomedical problems posed by action modes and drug localization in human pathology. SIMS microscopy should provide a new tool for metabolic radiotherapy by facilitating dose evaluation. The advent of high lateral resolution SIMS imaging (less than 0.1 microns) should open up new fields in biomedical investigation.     

Characterisation of P. falciparum antigenic determinants isolated from a genomic expression library by differential antibody screening.

A genomic expression library of P.falciparum has been differentially screened with a number of immune sera. The response of 9 clones to the various sera is presented, together with the DNA sequence encoding the epitopes. All but one clone are extremely A+T rich and unlike the other P.falciparum epitopes described, are not composed of amino acid repeats. One clone, which responds specifically with a protective serum, has been analysed in detail. The epitope is carried on a 160kd antigen which is transcribed from a single gene to give a protein expressed in all of the erythrocytic forms. DNA sequence of this clone reveals it to have more than one open reading frame, only one of which is transcribed in the blood stages. The possible significance of the other open readings frames is discussed.     

Abnormal electrophoretic mobility of spectrin tetramers in hereditary elliptocytosis

Summary Hereditary elliptocytosis (HE) is a genetically determined disorder of the red cell membrane. The main protein which composes the proteinaceous skeleton of the membrane is an elongated molecule named spectrin which is a heterodimer composed of two chains, and . In the membrane spectrin dimers are associated head-to-head to form tetrameric structures. We and other authors have reported that spectrin studied from many HE patients exhibited a dimer self-association defect (type I HE). A mutation in the head of the spectrin chain was mostly found in type I HE. We have previously described one of the three known spectrin pathological variants shown on mild tryptic digest pattern. This variant was characterized by the appearance of an abnormal 65,000-dalton peptide (Sp ^I/65). Using nondenaturating gel electrophoresis, we describe in this paper a triplicated pattern of the spectrin tetramer bands which is found in heterozygous HE cases displaying the 65,000-dalton variant. Study of a homozygous case allowed us to characterize the electrophoretic mobility of the abnormal symmetrical spectrin tetramer (₂ ^I/65-₂) and to study the correlation between the fraction of this abnormal symmetrical tetramer found in heterozygous patients and the amount of the 65,000-dalton peptide observed in spectrin tryptic digests.     

Chemical synthesis of a 7 kDa insect gonadotropic neurohormone

An original insect neurohormone of 65 residues was synthesized by the solid-phase methodology using t-Boc strategy and Boc-Val-PAM–resin. The purification, conducted by several steps of liquid chromatography having mass, polarity or charge as separative criteria, yielded the product with the correct molecular weight of 6922 Da determined by mass spectrometry. The synthetic peptide had both the same affinity for the antinative neurohormone serum and the same biological activity as the native neurohormone.     

Use of synthetic lethal mutants to clone and characterize a novel CTP synthetase gene in Saccharomyces cerevisiae

In the pyrimidine biosynthetic pathway, CTP synthetase catalyses the conversion of uridine 5-triphosphate (UTP) to cytidine 5-triphosphate (CTP). In the yeast Saccharomyces cerevisiae, the URA7 gene encoding this enzyme was previously shown to be nonessential for cell viability. The present paper describes the selection of synthetic lethal mutants in the CTP biosynthetic pathway that led us to clone a second gene, named URA8, which also encodes a CTP synthetase. Comparison of the predicted amino acid sequences of the products of URA7 and URA8 shows 78% identity. Deletion of the URA8 gene is viable in a haploid strain but simultaneous presence of null alleles both URA7 and URA8 is lethal. Based on the codon bias values for the two genes and the intracellular concentrations of CTP in strains deleted for one of the two genes, relative to the wild-type level, URA7 appears to be the major gene for CTP biosynthesis. Nevertheless, URA8 alone also allows yeast growth, at least under standard laboratory conditions.     

Identification of two human sperm populations using flow and image cytometry

Flow cytometric studies of human sperm from fertile men display a constant and characteristic bimodal nonartifactual DNA pattern confirming the existence of two distinct populations. The main population is represented by a peak followed by a shoulder (“marginal population”). The appearance of this marginal population fluctuates with either freezing and thawing or with Percoll gradient centrifugation. We have analyzed both the main and marginal sperm populations by flow cytometry after cell sorting, laser scanning cytometry, light microscopic evaluation, and their sensitivity to DNase digestion. We have observed that the marginal population detected in fertile men represents a sperm group altered in the nuclear condensation, yielding unstable chromatin which appears more stainable with propidium iodide. © 1994 Wiley-Liss, Inc.