IRE-Bubble PCR: A Rapid Method for Efficient and Representative Amplification of Human Genomic DNA Sequences from Complex Sources

A significant issue in the analysis of any genomic DNA segment is the generation of a unique set of short single-copy sequences that are representative of that region. In this report we describe a novel technique, IRE-bubble PCR, which was designed to amplify the human DNA content of somatic cell hybrids, YACs, cosmids, and λ phage and result in greater complexity and representation than standard inter-IRE, PCR. Here we demonstrate that IRE-bubble PCR is species specific and that it results in the generation of a product that is at least 10-fold more complex and representative than that produced by standard inter-IRE PCR. In addition, we have addressed the factors that contribute to the representation of the IRE-bubble PCR product and show how they may be used to further increase the complexity of this reaction. Finally, we have illustrated how the complexity and distribution of products generated by IRE-bubble PCR can be exploited and applied to FISH mapping and "chromosome painting" as well as to the generation of STSs targeted to specific chromosomal or subchromosomal regions.     

DNA sequence variation and phylogenetic relationships among strains of Pseudomonas syringae pv. syringae inferred from restriction site maps and restriction fragment length polymorphism.

We evaluated the restriction fragment length polymorphism of genomic DNA among 53 strains of the phytopathogenic bacterium Pseudomonas syringae pv. syringae. Twenty-nine strains were isolated from beans, and the rest were isolated from 11 other hosts. Southern blots of DNA digested with EcoRI or HindIII were hybridized to two random probes from a cosmid library of P. syringae pv. syringae and a hrp (hypersensitive reaction and pathogenicity) cluster cloned from P. syringae pv. syringae. The size of hybridizing fragments was determined, and a similarity matrix was constructed by comparing strains on a pairwise basis for the presence or absence of fragments. The proportion of shared fragments was then used to estimate sequence divergence. Dendrograms were produced by using the unweighted pair group method with averages and the neighbor-joining method. For the hrp region, BamHI, EcoRI, EcoRV, and HindIII restriction sites were mapped for six representative bean strains and used to construct EcoRI and HindIII restriction maps for all 30 strains pathogenic on beans. Restriction mapping revealed the presence of a 3-kb insertion in nine bean strains and a probable second insertion or deletion event on the left-hand side of the hrp cluster that biased estimates of nucleotide sequence divergence from fragment comparisons. This demonstrated that the determination of phylogenetic relationships among bacteria by using restriction fragment length polymorphism data requires mapping restriction sites to remove the effect of insertion or deletion events on the analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ecological impact of a fresh-water “reef kill” in Kaneohe Bay,Oahu, Hawaii

Storm floods on the night of December 31, 1987 reduced salinity to 15 in the surface waters of Kaneohe Bay, resulting in massive mortality of coral reef organisms in shallow water. A spectacular phytoplankton bloom occurred in the following weeks. Phytoplankton growth was stimulated by high concentrations of plant nutrients derived partially from dissolved material transported into the bay by flood runoff and partially by decomposition of marine organisms killed by the flood. Within two weeks of the storm, chlorophyll a concentrations reached 40 mg m^-3, one of the highest values ever reported. The extremely rapid growth rate of phytoplankton depleted dissolved plant nutrients, leading to a dramatic decline or crash of the phytoplankton population. Water quality parameters returned to values approaching the long-term average within 2 to 3 months. Corals, echinoderms, crustaceans and other creatures suffered extremely high rates of mortality in shallow water. Virtually all coral was killed to depths of 1–2m in the western and southern portions of the bay. Elimination of coral species intolerant to lowered salinity during these rare flood events leads to dominance by the coral Porites compressa. After a reef kill, this species can eventually regenerate new colonies from undifferentiated tissues within the dead perforate skeleton. Catastrophic flood disturbances in Kaneohe Bay are infrequent, probably occurring once every 20 to 50 years, but play an important role in determination of coral community structure. The last major fresh water reef kill occurred in 1965 when sewage was being discharged into Kaneohe Bay. Coral communities did not recover until after sewage abatement in 1979. Comparison between recovery rate after the two flood events suggests that coral reefs can recover quickly from natural disturbances, but not under polluted conditions.     

Mouse chromosome 1

Chair of Committee for Mouse Chromosome 1     

FINE STRUCTURE OF THE SPERMATOZOON OF HYDROIDES HEXAGONUS (ANNELIDA), WITH SPECIAL REFERENCE TO THE ACROSOMAL REGION

This paper describes in some detail the structure of the acrosomal region of the spermatozoon of Hydroides as a basis for subsequent papers which will deal with the structural changes which this region undergoes during fertilization. The material was osmium-fixed and mild centrifugation was used to aggregate the spermatozoa from collection to final embedding. The studies concern also the acrosomal regions of frozen-thawed sperm prepared by a method which previously had yielded extracts with egg membrane lytic activity. The plasma membrane closely envelops four readily recognizable regions of the spermatozoon: acrosomal, nuclear, mitochondrial, and flagellar. The acrosome consists of an acrosomal vesicle which is bounded by a single continuous membrane, and its periphery is distinguishable into inner, intermediate, and outer zones. The inner and intermediate zones form a pocket into which the narrowed apex of the nucleus intrudes. Granular material adjoins the inner surface of the acrosomal membrane, and this material is characteristically different for each zone. Centrally, the acrosomal vesicle is spanned by an acrosomal granule: its base is at the inner zone and its apex at the outer zone. The apex of the acrosomal granule flares out and touches the acrosomal membrane over a limited area. In this limited area the adjoining granular material of the outer zone is lacking. The acrosomal membrane of the inner zone is invaginated into about fifteen short tubules. The acrosomal membrane of the outer zone is closely surrounded by the plasma membrane. At the apex of the acrosomal region a small apical vesicle is sandwiched between the plasma membrane and the acrosomal membrane. Numerous frozen-thawed specimens and occasional specimens not so treated show acrosomal regions at the apex of which there is a well defined opening or orifice. Around the rim or lip of this orifice plasma and acrosomal membranes may even be fused into a continuum. The evidence indicates that the apical vesicle and the parts of the plasma and acrosomal membranes which surround it constitute a lid, and the rim of this lid constitutes a natural "fracture line" or rim of dehiscence. Should fracture occur, the lid would be removed and the acrosomal vesicle would be open to the exterior.     

Abrogation of the Fc gamma receptor IIA-mediated phagocytic signal by stem-loop Syk antisense oligonucleotides.

The role of Syk kinase in Fc gamma receptor (Fc gamma R) IIA-mediated phagocytosis was examined with two forms of antisense oligodeoxynucleotides (ODNs) designed to hybridize to human Syk mRNA. Monocytes were incubated with linear and stem-loop antisense ODNs targeted to Syk mRNA. When complexed with cationic liposomes, stem-loop Syk antisense ODN with phosphorothioate modification exhibited stability in fetal bovine and human serum. The stem-loop Syk antisense ODN at a concentration of 0.2 microM inhibited Fc gamma RIIA-mediated phagocytosis by 90% and completely eliminated Syk mRNA and protein in monocytes, whereas scrambled-control ODNs had no effect. The Syk antisense ODNs did not change beta-actin mRNA levels and Fc gamma RII cell-surface expression. In addition, stem-loop Syk antisense ODN inhibited Fc gamma RI and Fc gamma RIIIA-mediated phagocytosis. These data indicate the efficacy of stem-loop Syk antisense ODN for targeting and degrading Syk mRNA and protein and the importance of Syk kinase in Fc gamma receptor-mediated phagocytosis. Immunoblotting assay demonstrated that Fc gamma RII tyrosine phosphorylation after Fc gamma RII cross-linking did not change in the absence of Syk protein. These results indicate that Syk kinase is required for Fc gamma RIIA-mediated phagocytic signaling and that Fc gamma RII cross-linking leads to tyrosine phosphorylation of Fc gamma RII independent of Syk kinase.     

Influence of rhizodeposition under elevated CO2 on plant nutrition and soil organic matter

Atmospheric CO₂ concentrations can influence ecosystem carbon storage through net primary production (NPP), soil carbon storage, or both. In assessing the potential for carbon storage in terrestrial ecosystems under elevated CO₂, both NPP and processing of soil organic matter (SOM), as well as the multiple links between them, must be examined. Within this context, both the quantity and quality of carbon flux from roots to soil are important, since roots produce specialized compounds that enhance nutrient acquisition (affecting NPP), and since the flux of organic compounds from roots to soil fuels soil microbial activity (affecting processing of SOM).From the perspective of root physiology, a technique is described which uses genetically engineered bacteria to detect the distribution and amount of flux of particular compounds from single roots to non-sterile soils. Other experiments from several labs are noted which explore effects of elevated CO₂ on root acid phosphatase, phosphomonoesterase, and citrate production, all associated with phosphorus nutrition. From a soil perspective, effects of elevated CO₂ on the processing of SOM developed under a C4 grassland but planted with C3 California grassland species were examined under low (unamended) and high (amended with 20 g m^–2 NPK) nutrients; measurements of soil atmosphere 13C combined with soil respiration rates show that during vegetative growth in February, elevated CO₂ decreased respiration of carbon from C4 SOM in high nutrient soils but not in unamended soils.This emphasis on the impacts of carbon loss from roots on both NPP and SOM processing will be essential to understanding terrestrial ecosystem carbon storage under changing atmospheric CO₂ concentrations.Abbreviations SOM soil organic matter - NPP net primary productivity - NEP net ecosystem productivity - PNPP p-nitrophenyl phosphate     

Correspondence of environmental tolerances with leaf and branch attributes for six co-occurring species of broadleaf evergreen trees in northern California

 For the angiosperm dominants of northern California’s mixed evergreen forests, this study compares the display of photosynthetic tissue within leaves and along branches, and examines the correspondence between these morphological attributes and the known environmental tolerances of these species. Measurements were made on both sun and shade saplings of six species: Arbutus m e n z i e s i i (Ericaceae), C h r y s o l e p i s c h r y s o p h y l l a (Fagaceae), L i t h o c a r p u s d e n s i f l o r u s (Fagaceae), Quercus c h r y s o l e p i s (Fagaceae), Quercus w i s l i z e n i i (Fagaceae), and Umbellularia c a l i f o r n i c a (Lauraceae). All species had sclerophyllous leaves with thick epidermal walls, but species differed in leaf specific weight, thickness of mesophyll tissues and in the presence of a hypodermis, crystals, secretory idioblasts, epicuticular deposits, and trichomes. The leaves of Arbutus were 2 – 5 times larger than those of C h r y s o l e p i s, L i t h o c a r p u s and Umbellularia and 4 – 10 times larger than those of both Quercus species. Together with differences in branch architecture, these leaf traits divide the species into groups corresponding to environmental tolerances. Shade-tolerant C h r y s o l e p i s, L i t h o c a r p u s, and Umbellularia had longer leaf lifespans and less palisade tissue, leaf area, and crown mass per volume than the intermediate to intolerant Arbutus and Quercus. Having smaller leaves, Quercus branches had more branch mass per leaf area and per palisade volume than other species, whereas Arbutus had less than other species. These differences in display of photosynthetic tissue should contribute to greater growth for Quercus relative to the other species under high light and limited water, for Arbutus under high light and water availability, and for C h r y s o l e p i s, L i t h o c a r p u s, and Umbellularia under limiting light levels. Accepted: 22 March 1996     

Managed care. Origins,principles, and evolution.

Managed care has entered the lexicon of healthcare reform, but confusion and ignorance surround its meaning and purpose. It seeks to cut the costs of health care while maintaining its quality, but the evidence that it is able to achieve these aims is mixed. As well as raising awareness and understanding of the issues surrounding managed care, this series considers whether managed care is desirable for the NHS. Developed in the United States as a response to spiralling healthcare costs and dysfunctional fragmented services, managed care is not a discrete activity but a spectrum of activities carried out in a range of organisational settings. Due to its constantly changing nature, managed care is a slippery concept--but all its permutations have in common an attempt to influence and modify the behaviour and practice of doctors and other health professionals towards cost effective care. Whatever potential managed care may hold in this regard, careful appraisal of its implications is essential.