Structure of the sugar-phosphate moiety of lipid A from lipooligosaccharide of Neisseria meningitidis group B,strain BC5S No. 125

On the basis of chemical and NMR data the partial structure of lipid A from lipooligosaccharide (LOS) of Neisseria meningitidis group B, strain BC₅S No 125 was established. Lipid A consisted of disaccharide 2-deoxy-6-O-[2-deoxy-2-(3-hydroxytetradecanoylamino)--gluco-pyranosyl]-2-(3-hydroxytetradecanoylamino)--glucopyranose carrying the -(2-aminoethyl)pyrophosphate residue at 0–4 and the pyrophosphate or phosphate residue at 0–1. On hydrolysis of the acidic form of LOS with 1% acetic acid the substituent at 0–1 was practically completely removed whereas that at 0–4 was stable. The analogous hydrolysis of the Mg-salt of LOS was accompanied by splitting off the pyrophosphate linkage in the substituent at 0–4. Hydrolysis of LOS at pH 4.5 in the presence of SDS led mainly to a lipid A preparation retaining both pyrophosphate residues.Abbreviations KDO 2-keto-3-deoxyoctulosonic acid - LA-I, LA-II preparations of lipid A - LOS lipooligosaccharide - LOS-H⁺ the acidic form of LOS - OS oligosaccharide - TLC thin-layer chromatography - GLC-MS gas-liquid chromatography/mass spectrometry     

A conserved region in the sea urchin U1 snRNA promoter interacts with a developmentally regulated factor.

The expression of the sea urchin L. variegatus U1 snRNA gene is temporally regulated during embryogenesis. Using a microinjection assay we show that a region between 203 and 345 nts 5' of the gene is required for expression. There are four conserved regions between two sea urchin species in the 345 nts 5' to the U1 gene. One region, located at about -300, binds a protein factor which is present in blastula but not gastrula nuclei. Three other potential protein binding sites within the first 200 nts 5' to the gene have been identified using a mobility shift assay and/or DNase I footprinting. Two of these regions bind factors which are not developmentally regulated and one binds a factor which is developmentally regulated. It is likely that the factor which binds at -300 is involved in expression and developmental regulation of the sea urchin U1 snRNA gene.     

Isolation and culture of protoplasts of pigeonpea (Cajanus cajan L.)

Summary High yields of protoplasts were obtained from leaves of aseptically grown plants and calli originated from different explants, in several cultivars of Cajanus cajan L. The protoplasts divided to form cell clusters in modified KM 8p medium and developed to protocolonies after dilution with liquid Caboche's medium within three to four weeks of culture. The protocolonies proliferated to form green calli on solid Caboche's medium. No shoots or plants were obtained.Abbreviations BAP 6-benzylaminopurine - NAA -napthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Kin kinetin - Zea zeatin - Adn S adenine sulphate - GA ₃ gibberellic acid     

Construction and characterization of a region-specific microdissection library from human chromosome 2q35-q37.

A region-specific genomic library for human chromosome 2q35-q37 has been constructed using the microdissection and polymerase chain reaction-mediated linker-adaptor microcloning method. Twenty fragments from the chromosome region 2q35-q37 were dissected and a library consisting of 20,000 recombinant microclones was obtained. The insert size ranged between 50 and 800 bp, with a mean of approximately 270 bp. About 50-60% of the microclones contained unique sequences. The microdissection library has been demonstrated to derive from the dissected region 2q35-q37 by chromosome painting using the fluorescence in situ hybridization (FISH) technique. Southern blot analysis of the unique sequence microclones from the library showed that 54% (26/48) of the clones are of human origin and chromosome 2 specific. Four of these microclones have been further mapped to the 2q37 region by using a cell hybrid containing only 2q37. The unique sequence microclones have also been characterized for their insert size and the hybridizing genomic fragments cleaved with HindIII. As shown previously, these microclones will be useful in isolating corresponding yeast artificial chromosome (YAC) clones with large inserts for high-resolution physical mapping and also in screening cDNA libraries to isolate expressed gene sequences as candidate genes to facilitate search for the crucial genes underlying genetic diseases and specific forms of cancer assigned to the region.     

Protection of DNA damage by dietary restriction.

Dietary restriction is known to retard the aging processes and delay the onset of age-related neoplastic diseases. The mechanisms underlying these remarkable actions of nutritional intervention are not known in spite of recently intensified research efforts. However, the last couple of years' research on dietary restriction produced strong evidence indicating that its effective antiaging actions might be related to its ability to modulate free radical damage. In the present study, DNA damage and attenuation of the damage by dietary restriction were assessed by measuring 8-hydroxydeoxyguanosine 8-OH dG) in both nuclear DNA (nuDNA) and mitochondrial DNA (mitDNA) fractions. The data show that substantially more damage (approximately 15 times) occurred in mitDNA compared to nuDNA. More interestingly, the DNA damage was significantly attenuated in dietarily restricted rats.     

A domain of the hepadnavirus capsid protein is specifically required for DNA maturation and virus assembly

Mutations introduced into the capsid gene of duck hepatitis B virus (DHBV) were tested for their effects on viral DNA synthesis and assembly of enveloped viruses. Four classes of mutant phenotypes were observed among a series of deletions of covering the 3' end of the capsid open reading frame. Class I mutant capsids were able to support normal single-stranded and relaxed circular viral DNA synthesis; class II mutant capsids supported normal single-stranded DNA synthesis but not relaxed circular DNA synthesis; class III mutant capsids resembled class II capsids, but viral DNA synthesis was inhibited 5- to 10-fold; and class IV capsids were severely restricted in their ability to support viral DNA synthesis. Class I capsids were assembled into enveloped virions, but class II, III, and IV capsids were not. Viral DNA synthesized inside class II capsids was normal with respect to minus-strand DNA initiation, plus-strand DNA initiation, and circularization of the DNA, but plus strands failed to be elongated to mature 3-kb DNA. The results suggest that a function of the capsid protein specifically required for viral DNA maturation is also required for assembly of nucleocapsids into envelopes. Thus, class II mutants appear to be defective in the appearance of the "packaging signal" for virus assembly (J. Summers and W. Mason, Cell 29:403-415, 1982).     

Genetic analysis of a lactococcal plasmid replicon

Summary The sequence and genetic organization was determined of the 2508 by lactococcal portion of pFX2, which was derived from a crypticLactococcus lactis subsp.lactis plasmid and used as the basis for construction of a series of lactococcal vectors. A lactococcal plasmid plus origin and two replication protein-coding regions (repA andrepB) were located. RepA has a helix-turn-helix motif, a geometry typical of DNA-binding proteins. RepB shows a high degree of homology to the plasmid replication initiation proteins from other gram-positive bacteria andMycoplasma. The transcribed inverted repeat sequence betweenrepA andrepB could form an attenuator to regulate pFX2 replication. Upstream of theori site, and in a region which was non-essential for replication, a 215 by sequence identical to the staphylococcal plasmid pE194 and carrying the RS_A site was identified. The genetic organization of this lactococcal plasmid replicon shares significant similarity with pE194 group plasmids.     

A physiological role for DNA supercoiling in the anaerobic regulation of colicin gene expression

Summary The mechanism of anaerobic regulation of synthesis of colicins E1, E2, E3, K and D was studied. It was found that anaerobiosis significantly increases expression of the genes for colicins E1, E2, E3, K, and D. Experiments with novobiocin (a DNA gyrase inhibitor) showed that colicin synthesis in minicells and derepressed colicin synthesis in cells are dramatically reduced by relaxation of DNA supercoiling. A good correlation was observed between the levels of colicin synthesis and plasmid DNA supercoiling and the degree of aeration of the cultures. Thus, the regulation of colicin gene expression in response to a change in aeration appears to be mediated by environmentally induced variations in DNA supercoiling.     

Synergistic effect of glycine and bacteriocin release protein in the release of periplasmic protein in recombinantE. coli

Summary The synergistic effect of using mitomycin C-induced bacteriocin release protein (BRP) and glycine on cell growth, protein expression and release in a recombinant strain RR1 ofE. coli was investigated. An optimal combination of 50 ng/ml mitomycin C and 0.5% glycine concentration enhanced the release of periplasmic proteins from the cell into the fermentation broth without significantly affecting protein productivity. Under this optimal condition, the percentage of -amylase released into the broth increased from 7–13% to as much as 78%. The cell growth curve and low extracellular activity of the cytoplasmic protein -galactosidase show that there is no appreciable cell lysis.