Functional limits of oriP, the Epstein-Barr virus plasmid origin of replication.

The Epstein-Barr virus (EBV) genome contains two cis-acting elements which are required for stable extrachromosomal plasmid maintenance in latently infected cells. The first consists of 20 30-base-pair (bp) repeats, each of which contains a DNA-binding site for EBV nuclear antigen 1 (EBNA-1), the trans-acting factor required for plasmid persistence. The second element is composed of a 65-bp dyad symmetry, containing four EBNA-1-binding sites. Deletion mutants were constructed which reduce the number of EBNA-1-binding sites in the 30-bp repeats, alter the number of EBNA-1-binding sites in the dyad region, or truncate the dyad element. The effect of the deletion mutations on plasmid maintenance was examined by transfecting recombinant plasmids, containing both the mutated EBV sequences and a drug resistance marker, into D98-Raji cells. The plasmids were tested for their ability to generate drug-resistant D98-Raji cell colonies and their capacity to be maintained in an extrachromosomal form without undergoing extensive rearrangements. EBV plasmids with 12 or 15 copies of the 30-bp repeats were wild type in both assays. Plasmids with just two or six copies of these repeated elements failed to generate drug-resistant colonies at a normal level, and normal episomal plasmids were not detected in the resulting colonies. Rare colonies of cells resulting from transfection of these two- or six-copy mutants contained rearranged, episomal forms of the input plasmids. The rearrangements most often produced head-to-tail oligomers containing a minimum of eight 30-bp repeated elements. The rearranged plasmids were shown to be revertant for plasmid maintenance in that they yielded wild-type or greater numbers of drug-resistant colonies and persisted at the wild-type or a greater episomal copy number. By use of an EBV plasmid that contained no 30-bp elements, no revertants could be isolated. One to five copies of a synthetic linker corresponding to a consensus 30-bp repeated element inserted into a plasmid with no 30-bp elements now permitted the generation of oligomeric, episomal forms of the mutant test plasmid. These experiments demonstrate a requirement for a minimal number (six to eight copies) of the 30-bp repeated element. Deletions in the 65-bp dyad region had little or no effect upon the ability to generate enhanced numbers of drug-resistant D98-Raji colonies, indicating that the 30-bp repeated element is predominantly required for this phenotype.(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies with Differentially Labeled [11C]Cocaine, [11C]Norcocaine, [11C]Benzoylecgonine,and [11C]-and 4′-[18F]Fluorococaine to Probe the Extent to Which [11C]Cocaine Metabolites Contribute to PET Images of the Baboon Brain

Abstract: The psychostimulant drug of abuse, cocaine (benzoylecgonine methyl ester), is rapidly metabolized by cleavage of its two ester groups, to give benzoylecgonine (BE) and ecgonine methyl ester, and by N-demethylation, to give N-norcocaine (NC). The recent use of [N-methyl-¹¹CH₃]cocaine to image brain cocaine binding sites with positron emission tomography (PET) raises the question of whether PET images partially reflect the distribution and kinetics of labeled cocaine metabolites. We prepared [O-metty/-¹¹CH₃]cocaine by methylation of the sodium salt of BE with [¹¹C]CH₃l, and showed that PET baboon brain scans, as well as regional brain kinetics and plasma time-activity curves corrected for the presence of labeled metabolites, are nearly identical to those seen with [N-methyl-¹¹CH₃]cocaine. This strongly suggests that ¹¹C metabolites do not significantly affect PET images, because the metabolite pattern is different for the two labeled forms of cocaine. In particular, nearly half the ¹¹C in blood plasma at 30 min was [¹¹C]CO₂ when [N-methy/-¹¹CH₃]cocaine was administered, whereas [¹¹C]CO₂ was not formed from [O-methy/-¹¹CH₃]cocaine. Only a trace of [¹¹C]NC was detected in plasma after [O-methyl-¹¹CH₃]cocaine administration. Nearly identical brain PET data were also obtained when 4′-[N-methy/-¹¹CH₃]fluorococaine and 4′-[¹⁸F]fluoro-cocaine (prepared by nucleophilic aromatic substitution from [¹⁸F]fluoride-and 4′-nitrococaine) were compared with [N-methy/-¹¹CH₃]cocaine. In vitro assays with rat brain membranes showed that cocaine and 4′-fluoroco-caine were equipotent at the dopamine reuptake site, but that 4′-fluorococaine was about 100 times more potent at the 5-hydroxytryptamine reuptake site. The studies with positron-emitting 4′-fluorococaines thus support the lack of significance of labeled metabolites or of binding to 5-hydroxytryptamine reuptake sites to PET images taken with [N-methy/-¹¹CH₃]cocaine. [¹¹C]NC prepared by O-methylation of norbenzoylecgonine gave PET images with preferential uptake in striatum, but slower clearance from all brain regions than [O-methy/-¹¹CH₃]cocaine. [¹¹C]BE prepared by N-methylation of norbenzoylecgonine did not show brain uptake.     

Identifying plant functional types using floristic data bases: Ecological correlates of plant range size

Abstract. In an effort to identify ‘plant functional types’, the islands floras of Great Britain and Kríti (Crete, Greece) were examined separately for ecological correlates of plant range size. Plant functional types (PFTs) were defined here as categories into which plants could be grouped on the basis of attributes that predict greater or lesser sensitivity to ecological variability. Plant range size indicates commonness of a species and was assumed to be a proxy for ‘ecological flexibility’, i.e. species of larger range sizes can better withstand environmental change and differences than species of smaller range sizes. Using evolutionary comparative methods that account for the effect of taxonomic relatedness, both floras were investigated for the effects on range size of woodiness vs. non-woodiness, trees vs. shrubs, trees vs. herbs and shrubs vs. herbs. The British flora was examined additionally for the effects of wind- vs. non-wind-pollination, self vs. animal pollination and animal vs. non-animal fruit dispersal on range size. Two analyses showed significant effects on range size: for British species, trees had larger ranges than shrubs, and wind- pollinated species had larger ranges than non-wind-pollinated species. It is suggested that the lack of a similar pattern for shrubs and trees in Kríti is because the lower water availability of Kríti imbues shrubs with an ecophysiological advantage not relevant in plants of Great Britain. That trees have larger range sizes than shrubs in Great Britain is ascribed to the greater importance of competition for light when other factors are not at issue. The greater range of wind-pollinated than non-windpollinated species in Great Britain is postulated to be because both mutualists must be capable of invading new areas. This may be termed a ‘cost of mutualism’. In terms of PFTs, the results indicate that ‘life-form’ is too broad a classification category by which to differentiate relative sensitivity to environmental variability in Great Britain, in that there were significant differences in range size of trees and shrubs, but not between either of the two categories and herbs, or between woody and non-woody plants. Although pollination type may predict relative sensitivity to variation in Great Britain, dispersal type will not. Finally, differences between Great Britain and Kríti in relative range size patterns suggests that plant functional types may be specific to a region or set of conditions.     

The thermal biology of three southern African elephant-shrews

1. 1. The thermal characteristics of Petrodromus tetradactylus, Elephantulus intufi and E. brachyrhynchus were investigated and compared with other elephant-shrews that occur in the southern African subregion.

2. 2. E. intufi and E. brachyrhynchus appear to have lower than expected basal metabolic rates (1.1185 ± 0.1623 and 0.9649 ± 0.1638 ml O₂ g⁻¹ h⁻¹, respectively) and high, narrow thermoneutral zones, similar to other elephant-shrews investigated previously. In contrast P. tetradactylus has a basal metabolic rate (0.871 ± 0.027 ml O₂ g⁻¹ h⁻¹) close to expected for body mass, and a broad, low thermoneutral zone.

3. 3. The thermal biology of macroscelids is discussed in terms of their distribution, microhabitat and body size.

     

Three patterns of mitochondrial DNA nucleotide divergence in the meadow vole,Microtus pennsylvanicus

Summary The DNA sequence was determined for the cytochrome c oxidase II (COII), tRNA^Lys, and ATPase 8 genes from the mitochondrial genome of the meadow vole, Microtus pennsylvanicus. When compared to other rodents, three different patterns of evolutionary divergence were found. Nucleotide variation in tRNA^Lys is concentrated in the TC loop. Nucleotide variation in the COII gene in three genera of rodents (Microtus, Mus, Rattus) consists predominantly of transitions in the third base positions of codons. The predicted amino acid sequence in highly conserved (>92% similarity). Analysis of the ATPase 8 gene among four genera (Microtus, Cricetulus, Mus, Rattus) revealed more detectable transversions than transitions, many fixed first and second position mutations, and considerable amino acid divergence. The rate of nucleotide substitution at nonsynonymous sites in the ATPase 8 gene is 10 times the rate in the COII gene. In contrast, the estimated absolute mutation rate as determined by analysis of nucleotide substitutions at fourfold degenerate sites probably is the same for the two genes. The primary sequences of the ATPase 8 and COII peptides are constrained differently, but each peptide is conserved in terms of predicted secondary-level configuration.