Elimination of the non-specific binding of avidin to tissue sections

Summary A simple procedure is described for eliminating non-specific staining with avidin—peroxidase conjugates. Murine ovaries were embedded in either paraffin wax or epoxy resin and, after blocking endogenous peroxidase activity, were treated with 10 µg/ml biotinylatedPisum sativum agglutinin. Avidin—peroxidase conjugates (5 µg/ml), diluted in standard 0.05m tris-buffered saline, pH 7.6, containing 0.139m NaCl, produced considerable background coloration and intense mast cell staining in controls without the lectin. This background diminished as the ionic strength of the buffer was raised. At 0.125m Tris-buffered saline (containing 0.347m NaCl) the background was completely unstained, with elimination of all binding to mast cells and only minimal loss of specific lectin binding.     

Properties of the reaction center of the thermophilic purple photosynthetic bacterium Chromatium tepidum

Reaction centers were purified from the thermophilic purple sulfur photosynthetic bacterium Chromatium tepidum. The reaction center consists of four polypeptides L, M, H and C, whose apparent molecular masses were determined to be 25, 30, 34 and 44 kDa, respectively, by polyacrylamide gel electrophoresis. The heaviest peptide corresponds to tightly bound cytochrome. The tightly bound cytochrome c contains two types of heme, high-potential c-556 and low-potential c-553. The low-potential heme is able to be photooxidized at 77 K. The reaction center exhibits laser-flash-induced absorption changes and circular dichroism spectra similar to those observed in other purple photosynthetic bacteria. Whole cells contain both ubiquinone and menaquinone. Reaction centers contain only a single active quinone; chemical analysis showed this to be menaquinone. Reaction center complexes without the tightly bound cytochrome were also prepared. The near-infrared pigment absorption bands are red-shifted in reaction centers with cytochrome compared to those without cytochrome.     

Construction of an Unstable Ring-X Chromosome Bearing the Autosomal Dopa Decarboxylase Gene in Drosophila melanogaster and Analysis of Ddc Mosaics

An unstable Ring-X chromosome, Ddc⁺- Ring-X carrying a cloned Dopa decarboxylase (Ddc) encoding segment was constructed. The construction involved a double recombination event between the unstable Ring-X, R(1)w^vC and a Rod-X chromosome which contained a P-element mediated Ddc ⁺ insert. The resulting Ddc⁺-Ring-X chromosome behaves similarly to the parent chromosome with respect to somatic instability. The Ddc⁺-Ring-X chromosome was used to generate Ddc mosaics. Analyses of Ddc mosaics revealed that while there was no absolute requirement for the Ddc ⁺ expression in either the epidermis or the nervous system, very large mutant clones did affect the viability of the mosaic.     

DNA structures associated with autonomously replicating sequences from plants

DNA fragments capable of conferring autonomous replicating ability to plasmids inSaccharomyces cerevisiae were isolated from four different plant genomes and from the Ti plasmid ofAgrobacterium tumefaciens. The DNA structure of these autonomously replicating sequences (ARSs) as well as two from yeast were studied using retardation during polyacrylamide gel electrophoresis and computer analysis as measures of sequence-dependent DNA structures. Bent DNA was found to be associated with the ARS elements. An 11 bp ARS consensus sequence required for ARS function was also identified in the elements examined and was flanked by unusually straight structures which were rich in A+T content. These results show that the ARS elements from genomes of higher plants have structural and sequence features in common with ARS elements from yeast and higher animals.Supported by Grant 1RO1-GM41708-O1 from the National Institute of Health.     

The lyotropic liquid crystal properties of n-octyl 1-O-beta-D-glucopyranoside and related n-alkyl pyranosides

X-ray diffraction patterns have been obtained for the lyotropic phases of n-octyl 1-O-beta-D-glucopyranoside and the related n-heptyl, n-nonyl and n-decyl compounds with water. The octyl compound exhibits all three liquid crystal phases and forms a micellar solution with increasing solvation, when the crystal come into contact with water at room temperature. The X-ray diffraction patterns show a one-dimensional lamellar phase with [dx] = 28.4 A, a three-dimensional face-centered cubic phase with [a] = 51.2 A, and a two-dimensional hexagonal phase with [a] = [b] = 36.7 A. The micellar solution has a distribution pattern with a maximum at [dx] = 33.8 A. Crystals of the heptyl, nonyl and decyl derivatives form only the lamellar phases and the micellar solution on contact with water at room temperature.     

Turnover of Extracellular DNA in Eutrophic and Oligotrophic Freshwater Environments of Southwest Florida

The turnover of extracellular DNA was investigated in oligotrophic springs of the Crystal River and the eutrophic Medard Reservoir of southwest Florida. The Medard Reservoir possessed large populations of bacterioplankton and phytoplankton (6.8 × 10⁹ cells per liter and 28.6 μg of chlorophyll a per liter, respectively), while the Crystal River springs only contained a fraction of the microbial biomass found in the Medard Reservoir. Although dissolved DNA values were greater in the Medard Reservoir, higher rates of DNA removal resulted in similar extracellular DNA turnover times in both environments (9.62 ± 3.6 h in the Crystal River and 10.5 ± 2.1 h in the Medard Reservoir). These results indicate that regardless of trophic status or microbial standing stock, extracellular DNA turns over rapidly in subtropical planktonic freshwater environments. Therefore, recombinant DNA sequences from released genetically engineered microorganisms might not be expected to survive for long periods of time in freshwater planktonic environments.     

Functional expression of dihydropyridine-insensitive calcium channels during PC12 cell differentiation by nerve growth factor (NGF), oncogenic ras,or src tyrosine kinase

1. Recombinant retroviruses were used to introduce a temperature-sensitive v-src gene and oncogenic c-Ha-ras into PC12 cells, and stable cell lines expressing these genes were established. 2. As previously reported, expression of v-src (Alema et al., 1985) or c-Ha-ras (Noda et al., 1985) in PC12 cells results in neurite outgrowth resembling that induced by NGF. We report here that v-src but not oncogenic c-Ha-ras induces a stable morphologic neuronal differentiation similar to treatment with NGF. Oncogenic c-Ha-ras-induced neurite outgrowth is not stable with long-term culture, rather the cells revert to an undifferentiated morphology with altered cell cycle kinetics. 3. The stable neuronal phenotype induced by v-src and NGF is characterized by the functional expression of dihydropyridine-insensitive calcium currents.     

The cis-acting DNA sequences required in vivo for bacteriophage Mu helper-mediated transposition and packaging

The 37,000 bp double-stranded DNA genome of bacteriophage Mu behaves as a plaque-forming transposable element of Escherichia coli. We have defined the cis-acting DNA sequences required in vivo for transposition and packaging of the viral genome by monitoring the transposition and maturation of Mu DNA-containing pSC101 and pBR322 plasmids with an induced helper Mu prophage to provide the trans-acting functions. We found that nucleotides 1 to 54 of the Mu left end define an essential domain for transposition, and that sequences between nucleotides 126 and 203, and between 203 and 1,699, define two auxiliary domains that stimulate transposition in vivo. At the right extremity, the essential sequences for transposition require not more than the first 62 base pairs (bp), although the presence of sequences between 63 and 117 bp from the right end increases the transposition frequency about 15-fold in our system. Finally, we have delineated the pac recognition site for DNA maturation to nucleotides 32 to 54 of the Mu left end which reside inside of the first transposase binding site (L1) located between nucleotides 1–30. Thus, the transposase binding site and packaging domains of bacteriophage Mu DNA can be separated into two well-defined regions which do not appear to overlap.Abbreviations attL attachment site left - attR attachment site right - bp base pairs - Kb kilobase pair - nt nucleotide - Pu Purine - Py pyrimidine - Tn transposable element State University of New York, Downstate Medical Center, Brooklyn, NY 11204 USA