Assembly of transcriptionally active chromatin in vitro: a possible role for topoisomerase II

Some models of in vitro chromatin assembly suggest a biphasic molecular mechanism. The first phase, nucleosome formation, is comprised of the formation of histone-DNA complexes which mature into a canonical nucleosome structure. The second phase represents the process by which these nucleosomes become properly spaced with a regular periodicity on the DNA. In this report, we examine the role of DNA topoisomerases in the latter phase of chromatin assembly. To study this process, we use a Xenopus laevis cell-free extract, which assembles quantitative amounts of chromatin on circular DNA templates, and the type II topoisomerase-specific antitumor drugs VM-26 and endrofloxicin. Our results suggest that nucleosome formation is unaffected by the presence of VM-26 or endrofloxicin. However, periodic spacing of nucleosomes is inhibited significantly by these drugs. In the absence of proper chromatin assembly, circular DNA molecules are processed into nucleoprotein complexes which are transcribed poorly. Taken together, these results indicate that the antitumor drugs VM-26 and endrofloxicin influence gene expression indirectly by blocking the periodic spacing of nucleosomes.     

Some Properties of Extracellular Acetylxylan Esterase Produced by the Yeast Rhodotorula mucilaginosa

The red yeast Rhodotorula mucilaginosa produced an esterase that accumulated in the culture supernatant on induction with triacetin. The enzyme was specific for substrates bearing an O-acetyl group, but was relatively nonspecific for the rest of the molecule, which could consist of a phenol, a monosaccharide, a polysaccharide, or an aliphatic alcohol. The esterase was more active against acetylxylan and glucose beta-d-pentaacetate than were a number of esterases from plant and animal sources, when activities on 4-nitrophenyl acetate were compared. The enzyme exhibited Michaelis-Menten kinetics and was active over a broad pH range (5.5 to 9.2), with an optimum between pH 8 and 10. In addition, the enzyme retained its activity for 2 h at 55 degrees C. The yeast that produced the enzyme did not produce xylanase and, hence, is of interest for the production of acetylxylan esterase that is free of xylanolytic activity.     

ReducedS-adenosylmethionine: Protein-lysineN-methyltransferase activity (protein methylase III) in shiverer mutant mouse brain

Mice with the dysmyelinating mutation shiverer were studied by measuring the activity of two protein methylases and myelin marker enzymes in the brain. It was observed thatS-adenosylmethionine: protein-lysineN-methyltransferase (protein methylase III, EC. 2.1.1.43) activity is significantly reduced in phenotypically affected homozygous shiverer (shi/shi) mutant mouse brain compared to the unaffected heterozygous littermate brain. This reduction in enzyme activity is manifested mainly by reduced formation of trimethyllysine during the in vitro methylation of histone. In contrast, myelin marker enzymes such as 2,3-cyclic nucleotide 3-phosphohydrolase and 5-nucleotidase as well asS-adenosyl-methionine: protein-carboxylO-methyltransferase (protein methylase II, EC. 2.1.1.24) activities were not significantly affected in these strains of mice.     

Isolation and characterization of streptomycin-resistant mutants in Nicotiana plumbaginifolia

Summary Streptomycin-resistant colonies were isolated from protoplast cultures of haploid Nicotiana plumbaginifolia based on their ability to green in medium containing 1 mg/ml streptomycin sulfate. The frequency of resistant colonies was 0.9×10^–5 in nonmutagenized culture, and increased ten-fold following treatment of culture with 10 g/ml N-methyl-N-nitro-N-nitrosoguanidine. Of a total of 52 resistant clones isolated, 2 gave rise to haploid, 15 to diploid, and 3 to tetraploid plants upon transfer of calli to differentiation medium. Leaf-segment and protoplast assays showed that all diploid regenerates were resistant to streptomycin but sensitive to chloramphenicol, kanamycin, lincomycin, neomycin, and spectinomycin. Plants in most diploid clones were fertile and able to set seeds when self-fertilized and crossed reciprocally to wild-type plants. Inheritance of streptomycin resistance was studied in the diploid clones and, without exception, the resistance was transmitted maternally. Comparative studies of the ultrastructure of organelles and protein synthesis in isolated chloroplasts between wild-type and resistant clones in the presence of streptomycin suggest that streptomycin resistance is controlled by chloroplasts.     

Human esterase D gene: complete cDNA sequence,genomic structure,and application in the genetic diagnosis of human retinoblastoma

Summary The gene encoding human esterase D (EsD), a member of the nonspecific esterase family, is a useful genetic marker for retinoblastoma (RB) and Wilson's disease. Previously we identified a cDNA clone from this gene and determined its chromosomal location. In this report, we present the complete cDNA sequence of the human EsD gene. A long open reading frame encoded a predicted protein of 282 amino acids with molecular weight of 30 kD. A computer-assisted search of a protein sequence data base revealed homology with two other esterases, acetylcholinesterase of Torpedo and esterase-6 of Drosophila. Homologous region were centered around presumptive active sites, suggesting that the catalytic domains of the esterases are conserved during evolution. Three genomic clones of this gene were also isolated and characterized by restriction mapping. At least ten exons were distributed over a 35-kb (kilobase pair) region; each exon contained an average of 100 basepairs (bp). A polymorphic site for Apa I, located within an intron of the esterase D gene, can be used to identify chromosome 13 carrying defective RB alleles within retinoblastoma families.     

Mechanism of adhesion of Alysiella bovis to glass surfaces

Alysiella bovis adheres to surfaces by means of short, ruthenium red-staining, rod-like fimbriae. The fimbriae remain associated with the cell envelope of A. bovis, even when sonicated or exposed sequentially to toluene, Triton X-100, lysozyme, ribonuclease, and deoxyribonuclease. Adhesion of outer membrane-derived cell wall ghosts of A. bovis to glass was inhibited by IO4-, sodium dodecyl sulfate, urea, pronase, and trypsin. Protease treatment digested the fimbriae from the distal end, and exposure to sodium dodecyl sulfate depolymerized the fimbriae. Exposure of ghosts to 1% sodium dodecyl sulfate preferentially solubilized a 16,500-dalton protein which was subsequently purified by gel filtration and demonstrated to be a glycoprotein (ca. 17% carbohydrate). Antibodies raised against the 16,500-dalton glycoprotein agglutinated whole cells and inhibited adhesion of ghosts to glass.     

The Lipid Composition of Urodele Myelin Which Lacks Hydroxycerebroside and Hydroxysulfatide

The concentrations of cerebrosides and sulfatides were measured in the nervous systems of urodeles and related orders with a high performance liquid chromatographic technique. The peripheral and central nervous systems of all three urodele species, Necturus maculosis (mud puppy, a salamander), Notophthalmus viridescens (eastern red spot newt), and Desmognathus ochropheus (mountain salamander), were found to be completely devoid of alpha-hydroxy fatty acid-containing cerebrosides and sulfatides. All species of reptiles and fish classes close to urodeles contain these galactolipids. The levels of nonhydroxy fatty acid-containing cerebrosides and sulfatides are essentially similar in both urodeles and reptiles. Myelin isolated from Necturus spinal cord had a specific density of 1.07, lighter than mammalian myelin. Except for the absence of hydroxycerebrosides and hydroxysulfatides, the lipid composition of Necturus spinal cord myelin is essentially similar to that of frog and rat myelin. The fatty acids of nonhydroxycerebrosides are rich in monounsaturated homologs of C22-C25, and the sphingoid base consists of both sphinganine and sphingosine. Electron microscopic examination of the sciatic nerve showed that the general structure and interlamellar distances of salamander and newt myelin are identical to those of frog, chameleon, and rat. Necturus myelin, therefore, can be used as a model for the study of the functional and structural role of hydroxygalactolipids.     

Feeding and growth of larval herring,Clupea harengus,in relation to density of copepod nauplii

Synopsis Feeding and growth rates of 1–3 wk old herring larvae from four different stocks were compared in laboratory experiments (8°C). For most of the larval groups, feeding rate was saturated at nauplii (Acartia tonsa, nauplii stages 3–5) densities over 301^–1 (5 g d.w. 1^–1). Specific growth rate increased asymptotically with nauplii density, and reached about 6% d^–1 at densities over 120 nauplii 1^–1. The growth rates attained in the laboratory were similar to field measured growth rates of similarly aged herring larvae at comparable food densities. Since food particles were homogenously distributed in the laboratory tanks, patches of dense plankton concentrations are, thus, apparently not necessary for larval growth and survival in the sea. Growth efficiency differed between larval groups, with large sized larvae being the most efficient in transforming ingested matter into growth. The difference probably relates to different sizes rather than to the different geographical origins of the larvae.