A Link between ORC-Origin Binding Mechanisms and Origin Activation Time Revealed in Budding Yeast

Eukaryotic DNA replication origins are selected in G1-phase when the origin recognition complex (ORC) binds chromosomal positions and triggers molecular events culminating in the initiation of DNA replication (a.k.a. origin firing) during S-phase. Each chromosome uses multiple origins for its duplication, and each origin fires at a characteristic time during S-phase, creating a cell-type specific genome replication pattern relevant to differentiation and genome stability. It is unclear whether ORC-origin interactions are relevant to origin activation time. We applied a novel genome-wide strategy to classify origins in the model eukaryote Saccharomyces cerevisiae based on the types of molecular interactions used for ORC-origin binding. Specifically, origins were classified as DNA-dependent when the strength of ORC-origin binding in vivo could be explained by the affinity of ORC for origin DNA in vitro, and, conversely, as ‘chromatin-dependent’ when the ORC-DNA interaction in vitro was insufficient to explain the strength of ORC-origin binding in vivo. These two origin classes differed in terms of nucleosome architecture and dependence on origin-flanking sequences in plasmid replication assays, consistent with local features of chromatin promoting ORC binding at ‘chromatin-dependent’ origins. Finally, the ‘chromatin-dependent’ class was enriched for origins that fire early in S-phase, while the DNA-dependent class was enriched for later firing origins. Conversely, the latest firing origins showed a positive association with the ORC-origin DNA paradigm for normal levels of ORC binding, whereas the earliest firing origins did not. These data reveal a novel association between ORC-origin binding mechanisms and the regulation of origin activation time.     

Role of basidiospores as inocula of mycorrhizal fungi of birch

Summary Fruitbodies of sheathing mycorrhizal fungi collected under birch (Betula pendula andB. pubescens) were suspended over pots of soil and the resulting spore-supplemented soils were planted with sedlings ofB. pendula. Inocybe lacera, I. lanuginella, Hebeloma sacchariolens andH. leucosarx formed mycorrhizas readily.Lactarius pubescens andLeccinum roseofracta did not form mycorrhizas from basidiospore inocula, even after prolonged periods of seedling growth.Paxillus involutus gave equivocal results, perhaps because the soil was unsuitable for this species. Storage of the basidiospore-supplemented soils for 6 months in outdoor conditions or in a growth room at 18°C did not materially alter the results.The results are discussed in relation to the concept of mycorrhizal succession.     

Metal chelate affinity precipitation of RNA and purification of plasmid DNA

The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine `tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.     

Pentyl-4-yn-valproic acid enhances both spatial and avoidance learning, and attenuates age-related NCAM-mediated neuroplastic decline within the rat medial temporal lobe

2-N-Pentyl-4-pentynoic acid [pentyl-4-yn-valproic acid (VPA)] is an analogue of valproic acid that induces neuritogenesis and increases neural cell adhesion molecule (NCAM) prevalence in cultured neural cells. As memory consolidation involves synapse growth, aided by cell adhesion molecule function, we determined whether or not pentyl-4-yn-VPA had cognition-enhancing properties. Pentyl-4-yn-VPA (16-85 mg/kg) significantly improved water maze learning and task retention when given prior to each training session. Acute administration of pentyl-4-yn-VPA also influenced memory consolidation processes as, when given at 3 h post-passive avoidance training, the amnesia induced by scopolamine given 6 h post-training was prevented in a dose-dependent manner. Chronic administration of pentyl-4-yn-VPA (16.8 or 50.4 mg/kg) also significantly reduced escape latencies in the water maze task, 24 h following the last drug administration. This improved spatial learning was accompanied by enhanced neuroplasticity as the expression of NCAM polysialylated neurons in the infragranular zone of the dentate gyrus and in layer II of the perirhinal and piriform cortex was increased significantly following chronic drug treatment. The cognition-enhancing qualities of pentyl-4-yn-VPA, combined with its ability to attenuate the age-related loss of the NCAM polysialylation state, suggest that it may effectively slow the onset of cognitive decline.     

Study of the effects of temperature, pH, NaCl, and aw on the proteolytic and lipolytic activities of cheese-related lactic acid bacteria by quadratic response surface methodology

The individual and interactive effects of temperature, pH, NaCl, and a_w on the proteolytic and lipolytic activities of Lactobacillus delbrueckii subsp. bulgaricus B397, Lactococcus lactis subsp. lactis T12, and Lb. plantarum 2739 were studied by quadratic response surface methodology. The effects on enzyme activities depended on the interactions among the independent variables, type of activity, substrate and, especially, species. The proteinase activity of strains B397 and T12 was affected differently by pH as individual or interactive terms depending on the type of substrate _sl- or β-casein. The increase of NaCl concentration (2.5–7.5%) under cheese-like conditions had a negative effect on the proteinase activity of strain T12. The effect of NaCl was related to the corresponding decrease in a_w. Aminopeptidases N and A, iminopeptidase and endopeptidase of Lc. lactis subsp. lactis T12 were strongly inhibited by pH 5–6 and NaCl concentration higher than 3.75%. The negative effects of these independent variables was in several cases enhanced by their interaction and/or by the interaction with the lowest temperatures. In contrast, the same peptidases of Lb. plantarum 2739 retained a high activity under the very hostile environmental conditions. Iminopeptidase and especially endopeptidase activities of strain 2739 were stimulated slightly by NaCl at concentrations up to 5%. Lipase/esterase activity of Lb. delbrueckii subsp. bulgaricus B397 was markedly inhibited under cheese-like conditions.     

Phylogeny of Marine Bacillus Isolates from the Gulf of Mexico

The phylogeny of 11 pigmented, aerobic, spore-forming isolates from marine sources was studied. Forty-two biochemical characteristics were examined, and a 16S rDNA sequence was obtained for each isolate. In a phylogenetic tree based on 16S sequencing, four isolates (NRRL B-14850, NRRL B-14904, NRRL B-14907, and NRRL B-14908) clustered with B. subtilis and related organisms; NRRL B-14907 was closely related to B. amyloliquefaciens. NRRL B-14907 and NRRL B-14908 were phenotypically similar to B. amyloliquefaciens and B. pumilus, respectively. Three strains (NRRL B-14906, NRRL B-14910, and NRRL B-14911) clustered in a clade that included B. firmus, B. lentus, and B. megaterium. NRRL B-14910 was closely related phenotypically and phylogenetically to B. megaterium. NRRL B-14905 clustered with the mesophilic round spore-producing species, B. fusiformis and B. sphaericus; the isolate was more closely related to B. fusiformis. NRRL B-14905 displayed characteristics typical of the B. sphaericus-like organisms. NRRL B-14909 and NRRL B-14912 clustered with the Paenibacillus species and displayed characteristics typical of the genus. Only NRRL B-14851, an unusually thin rod that forms very small spores, may represent a new Bacillus species. Received: 8 December 1999 / Accepted: 14 February 2000     

Wrapping of genomic polydA.polydT tracts around nucleosome core particles.

Five human clones containing genomic regions of polydA have been isolated by their ability to form intermolecular triple helices with agarose cross-linked polyU. All of these clones contain Alu repetitive DNA sequences. End-labelled DNA fragments containing these sequences have been successfully reconstituted onto nucleosome core particles by salt exchange. The structure of these has been examined by digesting with DNase I, hydroxyl radicals or diethylpyrocarbonate. DNase I cleavage of the polydA tracts is poor in the free DNA but is markedly enhanced at certain positions when complexed with nucleosome cores. Phased digestion patterns are observed which continue through the (A)n blocks and reveal an average helical periodicity of about 10 base pairs. The distance between adjacent maxima varies between 8-12 base pairs, suggesting that the exact helical repeat is not necessarily constant. One fragment containing the sequence (TA)11T34 reveals a 12 base pair repeat within the (AT)n region. A pUC19 polylinker fragment containing a block of A69.T69 cloned into the Smal site could also be reconstituted onto nucleosome cores and reveals the same phased DNaseI digestion pattern. The DNase I cleavage pattern is not identical at each of the maxima, suggesting that the structural distortions imposed by the core particles are not constant along the DNA.     

Activation of human thymocytes via the 50KD T11 sheep erythrocyte binding protein induces the expression of interleukin 2 receptors on both T3+ and T3- populations

Recent studies have demonstrated that the 50KD T11 molecule is a surface component of a macrophage-independent alternative pathway of human T cell activation that is unrelated to the T3/Ti antigen-MHC receptor complex. Given the expression of T11 on all human thymocytes, it was of interest to determine whether they could be activated via this pathway. The triggering of T11 by monoclonal antibodies anti-T112 and anti-T113, directed at two unique epitopes on the molecule, induced IL 2 receptor expression on both T3+ and T3- thymocytes but did not induce IL 2 production. Consequently, in contrast to peripheral blood T cells, thymocytes did not proliferate in response to anti-T112 and anti-T113 in the absence of exogenous IL 2. These studies suggest that IL 2 receptor gene activation precedes IL 2 gene activation in T cell development. The ability of the alternative pathway of T cell activation to induce IL 2 receptor expression on T3- thymocytes implies that the T11 molecule may have an important role in early thymocyte ontogeny.