A dominant role for DNA secondary structure in forming hypersensitive structures in chromatin

Previous work has suggested that potential information in DNA secondary structure might be used by cells to define DNAase I- and S1-sensitive chromatin structures associated with promoter and terminator regions. To test this hypothesis, supercoiled pBR322 was cotransfected into L cells. For the majority of transfected clones supercoil-induced S1-sensitive sites in pure pBR322 DNA are also S1-sensitive in L-cell nuclei. These results suggest that the potential of certain DNA sequences to form specific secondary structures in chromatin can be a dominant characteristic. A recombinant chicken β^A-lobin supercoiled plasmid was reconstituted in vitro with histones. The reconstituted chromatin also retained the ability to form S1-sensitive sites. Evidence suggests that DNA sequences capable of forming S1-sensitive sites in supercoiled plasmids may bind nucleosomes poorly after reconstitution with histones.     

Cooperative alignment of nu bodies during chromosome replication in the presence of cycloheximide

50% of control DNA is resistant to staphylococcal nuclease after digestion in isolated nuclei, while only 25% of the labeled DNA made in the presence of cycloheximide is resistant to nuclease. Nevertheless, cycloheximide DNA is folded into normal chromosomal subunits as evidenced by the observation that it generates nuclese limit-digest DNA fragments that are indistinguishable from controls. These results indicate that cycloheximide chromatin is associated with half the number of normal nu bodies. These nu bodies are probably recycled from the parental chromosome. Partial nuclease digestion of cycloheximide chromatin reveals that a normal pattern of monomer and multimer DNA fragments is generated up to octamers. The data are consistent with the idea that in the presence of cycloheximide, recycled parental histones become cooperatively aligned along the daughter double helices.     

Variation among 41 Genotypes of Tomato (Lycopersicon esculentumMill.) for Crossability toL. peruvianum(L.) Mill.

Even with the aid of tissue culture, crosses betweenLycopersiconesculentum(E) andL. peruvianum(P) typically yield few progeny.To determine whether some E genotypes produce more progeny perfruit that others when crossed with P, 41 E genotypes were crossedwith pollen bulked from five P accessions. This first experiment(expt 1) was replicated over 2 years. In a second experiment(expt 2), differences among three genotypes each of E and P,and among individual plants within E genotypes were investigated.The E genotypes for expt 2 were chosen for relatively high andlow crossability based on results of expt 1. The P genotypesfor expt 2 were from different accessions than those used inexpt 1. For both experiments, the 15 largest ovules from eachripe fruit were cultured aseptically for 1 month. Out of 1228fruit, 753 hybrids were obtained. For expt 1, significant genotypeby year interactions were observed. Within each year, therewere significant differences among E genotypes for crossability.In expt 2, significant effects were found for E genotypes, butnot for interactions between E and P genotypes, P genotypes,nor plants within E genotypes. Moreover, general crossabilityfor E genotypes using bulked pollen (expt 1) was indicativeof general crossability with three P accessions not presentin the bulk (expt 2). Thus, selecting E genotypes of high crossabilityto P is the key to obtaining progeny for gene introgression.Rare production of ExP seed which was large and had brown seedcoats typical of E seed indicated strong selection pressureto maintain separate species, but gene exchange in nature maybe possible albeit at a low rate over long periods of time. Interspecific hybridization; Lycopersicon esculentum; Lycopersicon peruvianum; ovule culture; speciation     

Novel route to preparation of high purity lysoplasmenylethanolamine

A rapid and simple method has been developed for the preparation of highly purified lysoplasmenylethanolamine. The starting material, a phosphatidylethanolamine (PE) sample that contained a mixture of the 1, 2 diacyl- and 1-O-alkenyl-2-acyl forms was subjected to mild alkaline methanolysis for 20 min at room temperature. Addition of chloroform and water with vigorous mixing, but without acidification at this point, led to a preferential retention of the lysoplasmenylethanolamine in the alkaline aqueous phase and complete separation of the methyl esters into the chloroform phase. Neutralization of the alkaline phase with dilute acetic acid, followed by addition of chloroform, allowed recovery of the lysoplasmenylethanolamine in the chloroform phase in very high yields (75-80% based on vinyl ether content of starting material). On the other hand, a preparation of cholineglycerophospholipids enriched in plasmenylcholine, treated in exactly the same manner, gave a lysoplasmenylcholine that was not retained in the alkaline phase, but partitioned primarily into the chloroform-rich phase together with the methyl esters. Characterization of the purified lysoplasmenylethanolamine was achieved by thin-layer chromatography and compositional analysis. In addition, fast atom bombardment mass spectral analysis of the intact lysoplasmenylethanolamine together with gas chromatography-mass spectrometry of the dimethyl acetals derived from the 1-O-alkenyl chains allowed further proof of the structure and an assessment of the purity of this compound.     

Storage of glycoprotein in NCTR-Balb/C mouse. Lectin histochemistry, and biochemical studies.

A strain of Balb/C mice carrying a lysosomal storage disorder exhibits metabolic and phenotypic abnormalities similar to patients with sphingomyelin-cholesterol lipidoses type II (i.e., Niemann-Pick C and D). Their foamy cells, which belong to the reticuloendothelial system, stained intensely by periodate-Schiff (PAS) reagent and were resistant to predigestion with diastase. To identify the chemical nature of the PAS-positive storage material, we applied lectin histochemistry and biochemical methods. Paraffin embedded sections, and delipidated frozen tissue sections, were treated with biotinylated lectins and localized with avidin-biotin-peroxidase complex. Araldite-embedded semithin sections were incubated with biotinylated lectins followed by avidin-gold and were enhanced with silver. By both histochemical methods the affected foamy cells stained positively as follows: Concanavalia ensiformis agglutinin, Datura stramonium agglutinin, Griffonia simplicifolia-I, Lens culinaris agglutinin, peanut agglutinin, Ricinus communis agglutinin-I, wheat germ agglutinin (WGA), and succinylated-WGA. Biochemical analysis of liver extracts complemented the histochemical data and demonstrated accumulation of glycoproteins containing polylactosaminoglycans in affected mice. Our findings indicate that the storage material in NCTR-Balb/C mice is heterogeneous. The lipids that are extracted by organic solvents during the histologic preparations mask the occurrence of polylactosaminoglycan containing glycoproteins in native frozen sections.     

Structural elucidation of the O-antigenic polysaccharides from Escherichia coli O21 and the enteroaggregative Escherichia coli strain 105.

The structure of the O-antigen polysaccharide of the lipopolysaccharide from an enteroaggregative Escherichia coli (strain 105) has been elucidated, using primarily one-dimensional and two-dimensional NMR experiments. The sequence of residues was deduced with heteronuclear multiple-bond correlation and NOESY experiments. The structure of the repeating unit of the polysaccharide from the enteroaggregative E. coli is as follows:[sequence: see text] The structure of the O-antigen from enteroaggregative E. coli strain 105 was shown to be identical with that of E. coli O21 by sugar and methylation analyses as well as by 1H-NMR and 13C-NMR spectroscopy.     

Fast atom bombardment-mass spectrometric identification of molecular species of platelet-activating factor produced by stimulated human polymorphonuclear leukocytes

Fast atom bombardment mass spectrometry was used to identify molecular species of platelet-activating factor (PAF) produced by stimulated human neutrophilic polymorphonuclear leukocytes. Normal and reverse-phase high performance liquid chromatography were employed to separate the individual regions with PAF activity prior to mass spectrometric analysis. The following alkyl chain homologs of acetyl glyceryl ether phosphorylcholine (AGEPC) were found: C16:0, C17:0, C18:0 and C18:1. There was also evidence for the presence of the C15:0 homolog, as well as other species which have not yet been identified.