A study of variance in measurements of tank-treading frequency in populations of normal human red cells

The statistical variance in rheoscopic measurements of tank-treading frequency ( TTF ) of normal human red cells has been investigated and the sources of this variance analyzed. Experiments were designed to expose the influence of donor, the stability of a donor's cells over a 2-month period, storage time from venipuncture to testing, cell age, applied shear rate and variations in the rheoscope gap due to trial-to-trial manipulation. The results show that the TTF varies linearly with applied shear rate within the range 20 to 180 s-1 and is strongly dependent on cell age. The slope of the TTF characteristic is not significantly different in cells taken from different, but hematologically normal adults nor does it change significantly over time in a particular donor. TTF measurements may be useful in distinguishing rheologically abnormal cell populations which differ from normal cells by at least 10%.     

Strong coupling of plant and fungal community structure across western Amazonian rainforests

The Amazon basin harbors a diverse ecological community that has a critical role in the maintenance of the biosphere. Although plant and animal communities have received much attention, basic information is lacking for fungal or prokaryotic communities. This is despite the fact that recent ecological studies have suggested a prominent role for interactions with soil fungi in structuring the diversity and abundance of tropical rainforest trees. In this study, we characterize soil fungal communities across three major tropical forest types in the western Amazon basin (terra firme, seasonally flooded and white sand) using 454 pyrosequencing. Using these data, we examine the relationship between fungal diversity and tree species richness, and between fungal community composition and tree species composition, soil environment and spatial proximity. We find that the fungal community in these ecosystems is diverse, with high degrees of spatial variability related to forest type. We also find strong correlations between α- and β-diversity of soil fungi and trees. Both fungal and plant community β-diversity were also correlated with differences in environmental conditions. The correlation between plant and fungal richness was stronger in fungal lineages known for biotrophic strategies (for example, pathogens, mycorrhizas) compared with a lineage known primarily for saprotrophy (yeasts), suggesting that this coupling is, at least in part, due to direct plant–fungal interactions. These data provide a much-needed look at an understudied dimension of the biota in an important ecosystem and supports the hypothesis that fungal communities are involved in the regulation of tropical tree diversity.     

Insights into mutagenesis using Escherichia coli chromosomal lacZ strains that enable detection of a wide spectrum of mutational events

Strand misalignments at DNA repeats during replication are implicated in mutational hotspots. To study these events, we have generated strains carrying mutations in the Escherichia coli chromosomal lacZ gene that revert via deletion of a short duplicated sequence or by template switching within imperfect inverted repeat (quasipalindrome, QP) sequences. Using these strains, we demonstrate that mutation of the distal repeat of a quasipalindrome, with respect to replication fork movement, is about 10-fold higher than the proximal repeat, consistent with more common template switching on the leading strand. The leading strand bias was lost in the absence of exonucleases I and VII, suggesting that it results from more efficient suppression of template switching by 3' exonucleases targeted to the lagging strand. The loss of 3' exonucleases has no effect on strand misalignment at direct repeats to produce deletion. To compare these events to other mutations, we have reengineered reporters (designed by Cupples and Miller 1989) that detect specific base substitutions or frameshifts in lacZ with the reverting lacZ locus on the chromosome rather than an F' element. This set allows rapid screening of potential mutagens, environmental conditions, or genetic loci for effects on a broad set of mutational events. We found that hydroxyurea (HU), which depletes dNTP pools, slightly elevated templated mutations at inverted repeats but had no effect on deletions, simple frameshifts, or base substitutions. Mutations in nucleotide diphosphate kinase, ndk, significantly elevated simple mutations but had little effect on the templated class. Zebularine, a cytosine analog, elevated all classes.     

Crossing over between regions of limited homology in Escherichia coli. RecA-dependent and RecA-independent pathways

We have developed an assay for intermolecular crossing over between circular plasmids carrying variable amounts of homology. Screens of Escherichia coli mutants demonstrated that known recombination functions can only partially account for the observed recombination. Recombination rates increased three to four orders of magnitude as homology rose from 25 to 411 bp. Loss of recA blocked most recombination; however, RecA-independent crossing over predominated at 25 bp and could be detected at all homology lengths. Products of recA-independent recombination were reciprocal in nature. This suggests that RecA-independent recombination may involve a true break-and-join mechanism, but the genetic basis for this mechanism remains unknown. RecA-dependent crossing over occurred primarily by the RecF pathway but considerable recombination occurred independent of both RecF and RecBCD. In many respects, the genetic dependence of RecA-dependent crossing over resembled that reported for single-strand gap repair. Surprisingly, ruvC mutants, in both recA(+) and recA mutant backgrounds, scored as hyperrecombinational. This may occur because RuvC preferentially resolves Holliday junction intermediates, critical to both RecA-dependent and RecA-independent mechanisms, to the noncrossover configuration. Levels of crossing over were increased by defects in DnaB helicase and by oxidative damage, showing that damaged DNA or stalled replication can initiate genetic recombination.     

A bacterial G protein-mediated response to replication arrest

To define factors in E. coli promoting survival to replication fork stress, we isolated insertion mutants sensitive to replication inhibitors. One insertion caused partial loss of the universally conserved GTPase, obgE/yhbZ gene. Although obgE is essential for growth, our insertion allele supported viability until challenged with various replication inhibitors. A mutation designed to negate the GTPase activity of the protein produced similar phenotypes, but was genetically dominant. Synergistic genetic interactions with recA and recB suggested that chromosome breaks and regressed forks accumulate in obgE mutants. Mutants in obgE also exhibited asynchronous overreplication during normal growth, as revealed by flow cytometry. ObgE overexpression caused SeqA foci, normally localized to replication forks, to spread extensively within the cell. We propose that ObgE defines a pathway analogous to the replication checkpoint response of eukaryotes and acts in a complementary way to the RecA-dependent SOS response to promote bacterial cell survival to replication fork arrest.     

Concerted transpositions of mobile genetic elements coupled with fitness changes in Drosophila melanogaster

In an inbred low-activity (LA) strain of Drosophila melanogaster with a low level of fitness and a complex of inadaptive characters, in situ hybridization reveals an invariant pattern of distribution of three copia-like elements (mdg-1, mdg-3, and copia). Rare, spontaneous, multiple transpositions of mobile elements in the LA strain were shown to be coupled with a drastic increase of fitness. A changed pattern of various types of mobile elements was also observed on selecting the LA strain for higher fitness. High-fitness strains show transpositions of mobile elements to definite chromosomal sites ("hot spots"). Concerted changes in the location of three different mobile elements were found to be coupled with an increase of fitness. The mdg-1 distribution patterns were also examined in two low-fitness strains independently selected from the high-fitness ones. Fitness decrease was accompanied by mdg-1 excision from the hot spots of their location usually detected in the high-fitness strains. The results suggest the existence of a system of adaptive transpositions of mobile elements that takes part in fitness control.      

Mutational Analysis of the RecJ Exonuclease of Escherichia coli: Identification of Phosphoesterase Motifs

The recJ gene, identified in Escherichia coli, encodes a Mg(+2)-dependent 5'-to-3' exonuclease with high specificity for single-strand DNA. Genetic and biochemical experiments implicate RecJ exonuclease in homologous recombination, base excision, and methyl-directed mismatch repair. Genes encoding proteins with strong similarities to RecJ have been found in every eubacterial genome sequenced to date, with the exception of Mycoplasma and Mycobacterium tuberculosis. Multiple genes encoding proteins similar to RecJ are found in some eubacteria, including Bacillus and Helicobacter, and in the archaea. Among this divergent set of sequences, seven conserved motifs emerge. We demonstrate here that amino acids within six of these motifs are essential for both the biochemical and genetic functions of E. coli RecJ. These motifs may define interactions with Mg(2+) ions or substrate DNA. A large family of proteins more distantly related to RecJ is present in archaea, eubacteria, and eukaryotes, including a hypothetical protein in the MgPa adhesin operon of Mycoplasma, a domain of putative polyA polymerases in Synechocystis and Aquifex, PRUNE of Drosophila, and an exopolyphosphatase (PPX1) of Saccharomyces cereviseae. Because these six RecJ motifs are shared between exonucleases and exopolyphosphatases, they may constitute an ancient phosphoesterase domain now found in all kingdoms of life.     

New observations and reinterpretation on the enigmatic taxon Colombitherium (?Pyrotheria,Mammalia) from Colombia

Abstract: The controversial taxon Colombitherium tolimense (Mammalia) (probably Late Eocene in age) from Colombia, although known for nearly 40 years, still bears much mystery. Aside from the problematic ordinal attribution of the holotype and only specimen, its determination as an upper or lower jaw remains a highly debated issue. New observations include the presence of a contact facet on the distal face of the most posterior tooth, which indicates that the fragmentary jaw preserves three premolars and two molars; the M3, unpreserved but present, being most probably reduced. This new interpretation completely fits the morphology of the teeth. Furthermore, the shape of these latter and the deeper wear encompassed by their lingual part relative to the labial one is typical of upper dentition. This is in agreement with the internal curving of the roots of the anterior premolars and with several other arguments that lead interpreting the holotype of C. tolimense as a maxillary bearing P2‐M2. This new interpretation deepens the morphological gap between Colombitherium and other pyrotherians (except Proticia) and challenges further its referral to Pyrotheria. The peculiar morphology of Colombitherium relative to other pyrotherians is indeed striking. In fact, Colombitherium has nothing in common with pyrotherians but bilophodont cheek teeth, a feature largely widespread in placental mammals. It is here referred to ?Pyrotheria until additional evidence of its relationships is known. Associated with the putative removal of Proticia from Pyrotheria as argued by some authors, the hypothetical removal of Colombitherium from the order would adjust the widely accepted assumption that the pyrotherian bilophodont cheek teeth originated from bunodont cheek teeth. It would also make an origin from lophodont forms plausible. This in turn would have critical relevance, especially to the hypothesis that pyrotherians are notoungulates.