Epstein-Barr virus recombinants from overlapping cosmid fragments

Five overlapping type 1 Epstein-Barr virus (EBV) DNA fragments constituting a complete replication- and transformation-competent genome were cloned into cosmids and transfected together into P3HR-1 cells, along with a plasmid encoding the Z immediate-early activator of EBV replication. P3HR-1 cells harbor a type 2 EBV which is unable to transform primary B lymphocytes because of a deletion of DNA encoding EBNA LP and EBNA 2, but the P3HR-1 EBV can provide replication functions in trans and can recombine with the transfected cosmids. EBV recombinants which have the type 1 EBNA LP and 2 genes from the transfected EcoRI-A cosmid DNA were selectively and clonally recovered by exploiting the unique ability of the recombinants to transform primary B lymphocytes into lymphoblastoid cell lines. PCR and immunoblot analyses for seven distinguishing markers of the type 1 transfected DNAs identified cell lines infected with EBV recombinants which had incorporated EBV DNA fragments beyond the transformation marker-rescuing EcoRI-A fragment. Approximately 10% of the transforming virus recombinants had markers mapping at 7, 46 to 52, 93 to 100, 108 to 110, 122, and 152 kbp from the 172-kbp transfected genome. These recombinants probably result from recombination among the transfected cosmid-cloned EBV DNA fragments. The one recombinant virus examined in detail by Southern blot analysis has all the polymorphisms characteristic of the transfected type 1 cosmid DNA and none characteristic of the type 2 P3HR-1 EBV DNA. This recombinant was wild type in primary B-lymphocyte infection, growth transformation, and lytic replication. Overall, the type 1 EBNA 3A gene was incorporated into 26% of the transformation marker-rescued recombinants, a frequency which was considerably higher than that observed in previous experiments with two-cosmid EBV DNA cotransfections into P3HR-1 cells (B. Tomkinson and E. Kieff, J. Virol. 66:780-789, 1992). Of the recombinants which had incorporated the marker-rescuing cosmid DNA fragment and the fragment encoding the type 1 EBNA 3A gene, most had incorporated markers from at least two other transfected cosmid DNA fragments, indicating a propensity for multiple homologous recombinations. The frequency of incorporation of the nonselected transfected type 1 EBNA 3C gene, which is near the end of two of the transfected cosmids, was 26% overall, versus 3% in previous experiments using transfections with two EBV DNA cosmids. In contrast, the frequency of incorporation of a 12-kb EBV DNA deletion which was near the end of two of the transfected cosmids was only 13%.(ABSTRACT TRUNCATED AT 400 WORDS)     

On the dynamics of the electric field induced breakdown in lipidmembranes

Electric field methods are powerful tools for cell characterization and manipulation. Important biotechnological applications, e.g., electrofusion of cells or electroinjection of macromolecules into living cells, rely on membrane breakdown. Despite the widespread use of these techniques, the process of membrane breakdown is poorly understood: a better understanding can be expected to lead to higher efficiencies. The present study used planar lipid bilayers in order to limit the number of experimental parameters. Membrane rupture was induced by careful application of short electric field pulses: measurement of the subsequent increase in membrane conductivity with time permitted the underlying mechanism to be characterized. The initial process of pore formation starts a few μs after the onset of the pulse, and the ensuing breakdown of the entire lipid membrane occurs within about a millisecond (pore widening velocity of a few cm/s). The kinetics of the pore-opening process could be influenced qualitatively by adding surfactants and lipid-attached macromolecules, respectively. Changing the effective mass per unit area of membrane, which could be brought about by using known percentages of lipids with covalently bound, hydrophilic polymers, allowed a model for the pore widening process to be quantitatively tested     

Setting the record straight.

Replies to comments by H. W. Marsh and L. A. Roche (see record 1997-43129-003) on the author's article (Educational Policy Analysis Archives, 1997) on student evaluation and academic freedom. The author expresses his dismay that Marsh and Roche misrepresented (not misinterpreted) the article without documenting their misrepresentation. A documented response to this undocumented misrepresentation is provided. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Recent thinking about the concept of alcohol dependence

This paper reviews past definitions of alcohol dependence as well as that presently adopted in the 4th edition of the Diagnostic and Statistical Manual of the American Psychiatric Association. The role of alcohol consumption in diagnostic criteria and the measurement of various dimensions of drinking (frequency, usual quantity per occasion, overall volume) are also discussed. Reliable criteria for identifying both alcohol abuse and dependence are available. Methodology for assessment of different dimensions of drinking behavior also exists. Epidemiologists examining the role of alcohol in health problems should consider the diagnostic category and the dimensions of drinking behavior that are important to their study. Measurements should be adopted in accordance with this assessment.     

Verification of ANSI C37.90.1 SWC test generator outputs

Awareness of fast transient events in the electrical power environment has increased with the release of related test standards (ANSI C37.90.1 and IEC 801-4) and with increased usage of solid state circuits in utility equipment. Reliable measurement of the fast transient test waveforms and repeatable performance of test setups are persistent problems for equipment manufacturers and users. The construction, characterization, and usage of two high voltage test dividers for measurement of fast transient test waveforms are described. Measurements on a C37.90.1 (SWC) test generator are presented along with tutorial background. Proposals for changes to the ANSI C37.90.1 specification are included     

Comparison of smoothness evaluation devices for power cablecompounds

A variety of data from different devices has been generated on the smoothness of shield products, but no direct comparison of compound smoothness for the devices themselves has been made. The purpose of this study was to compare four different methods for evaluating the smoothness of power cable compounds. The methods compared include: image analysis, laser scanning, mechanical profilometry and photomicroscopy with visual rating. Extruded tape samples of conductor shields, filled insulations and strippable shields were evaluated     

A bidding decision index for construction contractors

Results from applying the model on a sample of contractors, the majority of whom were international and operating in Egypt, reinforces the credibility of the developed methodology, claim the authors.