Dual fluorescent protein reporters for studying cell behaviors in vivo

Fluorescent proteins (FPs) are useful tools for visualizing live cells and their behaviors. Protein domains that mediate subcellular localization have been fused to FPs to highlight cellular structures. FPs fused with histone H2B incorporate into chromatin allowing visualization of nuclear events. FPs fused to a glycosylphosphatidylinositol anchor signal sequence label the plasma membrane, highlighting cellular shape. Thus, a reporter gene containing both types of FP fusions would allow for effective monitoring of cell shape, movement, mitotic stage, apoptosis, and other cellular activities. Here, we report a binary color‐coding system using four differently colored FP reporters that generates 16 distinct color codes to label the nuclei and plasma membranes of live cells in culture and in transgenic mice. As an initial test of this system in vivo, the promoter of the human Ubiquitin C (UBC) gene was used to widely express one of the color‐code reporters. Widespread expression of the reporter was attained in embryos; however, both male and female transgenic mice were infertile. In contrast, the promoter of the mouse Oct4/Pou5f1 gene linked to two different color‐code reporters specifically labeled blastocysts, primordial germ cells, and postnatal germ cells, and these mice were fertile. Time‐lapse movies of fluorescently‐labeled primordial germs cells demonstrate the utility of the color‐code system to visualize cell behaviors. This set of new FP reporters should be a useful tool for labeling distinct cell populations and studying their behaviors in complex tissues in vivo. genesis 47:708–717, 2009. © 2009 Wiley‐Liss, Inc.     

Intrinsic effects of species on leaf litter and root decomposition: a comparison of temperate grasses from North and South America

A simple model of water uptake by vegetation is used to aid the discrimination of plant water sources determined with isotope data. In the model, water extracted from different soil depths depends on the leaf–soil potential difference, a root distribution function and a lumped hydraulic conductance parameter. Measurements of plant transpiration rate, and soil and leaf water potentials are used to estimate the value of the conductance parameter. Isotopic ratios in soil water and xylem are then used to constrain the root distribution. The model is applied to field measurements of transpiration, leaf water potential and ¹⁸O composition of xylem water on Corymbia clarksoniana, Lophostemon suaveolens, Eucalpytus platyphylla and Melaleuca viridiflora, and soil water potential and ¹⁸O composition of soil water to 8.5 m depth, in an open woodland community, Pioneer Valley, North Queensland. Estimates of the water uptake from various depths below the surface are determined for each species. At the time of sampling, the proportion of groundwater extracted by the trees ranged from 100% for C. clarksoniana to <15% for L. suaveolens and E. platyphylla. The advantages of the model over the traditional approach to determining sources of water used by plants using isotope methods are that it: (1) permits more quantitative assessments of the proportion of water sourced from different depths, (2) can deal with gradational soil water isotope profiles (rather than requiring distinct values for end-members), and (3) incorporates additional data on plant water potentials and is based on simple plant physiological processes.     

Parasites in a biodiversity hotspot: a survey of hematozoa and a molecular phylogenetic analysis of Plasmodium in New Guinea skinks

A sample of 204 skinks (Squamata: Scincidae) from 10 genera representing 24 species were collected from 10 different localities in New Guinea and examined for blood parasites. Hemogregarines, trypanosomes, microfilarial worms, and 8 infections showing 2 distinct morphological types of malaria parasites (Plasmodium sp.) were observed. Molecular sequence data, in the form of mitochondrial cytochrome b sequences from the Plasmodium infections, showed 2 distinct clades of parasites, 1 in Sphenomorphus jobiense hosts and 1 in Emoia spp., which correspond to the 2 morphotypes. There was substantial genetic variation between the 2 clades, as well as within the clade of Emoia parasites. Nearly half of the skinks sampled had green blood pigmentation, resulting from the presence of biliverdin in the plasma; however, only 1 of these lizards was infected with Plasmodium sp. and only 2 had any blood parasites. These preliminary results suggest a high degree of phylogenetic diversity but a very low prevalence of Plasmodium spp. infections in the skinks of this globally important biodiversity hot spot.     

Toxicity of Clostridium botulinum type E neurotoxin to Great Lakes fish: implications for avian botulism

Since 1999, large-scale mortalities of fish-eating birds have been observed on the Great Lakes, and more specifically on Lake Erie. Type E botulism has been established as the primary cause of death. The mechanism of type E botulism exposure in fish-eating birds is unclear. Given that these birds are thought to eat live fish exclusively, it seems likely that their prey play a key role in the process, but the role of fish as potential transport vectors of botulinum neurotoxin type E (BoNT/E) to birds has not been adequately investigated. Between June 2003 and April 2004 a methodological model for exposing fish to Clostridium botulinum was developed and used to compare the sensitivity of rainbow trout (Oncorhynchus mykiss), round goby (Neogobius melanostomas), walleye (Stizostedion vitreum), and yellow perch (Perca flavescens) to four doses (0, 800, 1,500, and 4,000 Mouse Lethal Doses) of Clostridium botulinum type E neurotoxin. Each fish species expressed unique changes in both behavior and skin pigmentation prior to death. Yellow perch survived significantly longer (P < 0.05) than the three other species at all toxin treatments. Results of this study suggest that live fish can represent a significant vector for transfer of BoNT/E to birds.     

Differential selective pressures on the merozoite surface protein 2 locus of Plasmodium falciparum in a low endemic area

195 Plasmodium falciparum merozoite surface protein-2 alleles collected in Tak Province, Thailand, in 1996 and 2006 revealed extremely limited sequence polymorphism except in the variable (V) region, which defines the two allelic families 3D7 and FC27. This pattern is most easily explained by repeated inter-allelic gene conversion events homogenizing alleles outside the V region. Comparison of synonymous and nonsynonymous differences in V regions within allelic families supported the hypothesis that amino acid sequence polymorphism in this region is selectively favored. The pattern of sequence differentiation supported the hypothesis that repeats in the V region have evolved by concerted evolution in the 3D7 family but not in the FC27 family. In the FC27 family two alleles of relatively high frequency were the most common V-region alleles in both 1996 and 2006, while 3D7 alleles constituted a significantly greater proportion of the sequences collected in 2006 (56.1%) than of those collected in 1996 (28.9%). These changes in the frequencies of 3D7 alleles may reflect increased intensity of selection on the P. falciparum population in Thailand as a result of effective control measures that have sharply reduced the incidence of malaria infection.     

Incongruence in the pattern and timing of intra-specific diversification in bronze frogs and bullfrogs (Ranidae)

We compare patterns of lineage divergence in mitochondrial DNA (mtDNA) sequences of two protein-encoding mitochondrial genes (cyt b and ND2) in two ecologically similar, co-distributed, and closely related ranid frogs (Rana clamitans and Rana catesbeiana), that are geographically widespread, and frequently syntopic. We identified three lineages in R. clamitans, separated by 0.5% to 2.1% net corrected sequence divergence, comparable to two R. catesbeiana lineages separated by 0.6%. The geographic pattern of lineage distribution differed notably between the two species. In R. clamitans, we found a Coastal Plain-Appalachian (CPA) lineage restricted to south and east of the Appalachian Mountains and a widespread lineage that encompassing nearly all the sampled range. A third distinct and divergent lineage was detected in one location in the southwest portion of the range (Louisiana). This pattern contrasts with the east-west pattern in R. catesbeiana, and reflects possible differences in refugial dynamics and patterns of range expansion. Although both species have undergone range expansion and population growth, coalescent reconstruction of N(e) reflects larger lineages but more recent divergence in R. clamitans relative to R. catesbeiana, reflecting significant differences in population history or divergent patterns of molecular evolution at mtDNA.     

Genome size reduction in the chicken has involved massive loss of ancestral protein-coding genes

Both mean genomes size and the variance in genome size among species are smaller on average in birds (class Aves) than in the other tetrapod classes. In order to test whether loss of protein-coding genes has contributed to genome size reduction in birds, we compared the chicken genome and five mammalian genomes. Numbers of members (paralogs) were significantly lower in the chicken gene families than in the corresponding mammalian families. Phylogenetic analyses of chicken, mammal, and fish paralogs supported the hypothesis that chicken-specific loss of paralogs occurred much more frequently than mammal-specific gene duplications. Moreover, the phylogenetic analyses supported the hypothesis that a substantial majority of the paralogs lost in chicken originated from duplications prior to the most recent common ancestor of tetrapods and bony fishes. In addition to loss of paralogs, numerous gene families present in the mammalian genomes were missing in the chicken genome; over 1,000 of these families were found in bony fishes, implying presence of the family in the tetrapod ancestor. In the set of families with more members on average in the mammals than in the chicken, immune system function was associated with a greater degree of gene family size reduction in the chicken, consistent with other evidence that immune system gene families have become particularly compact in birds.