The ultrastructure of the germanium in some Rhabdocoela

Two Daphnia clones were isolated from different day depths during the period of diel vertical migration and were tested for their life-history responses to a fish exudate released by juvenile perch. Animals were exposed to fish exudate every 24 or 48 h. Both clones responded to the exudate by exhibiting earlier maturation and larger sizes of first clutches, which resulted in higher rates of population increase. Also, neonates were smaller in the presence of the exudate. It was found that the clone isolated from a deeper day depth (migrating clone) was less sensitive to the exudate than the clone isolated from the surface waters, which was presumed to be non-migrating. The non-migrating clone responded by having smaller neonate sizes and smaller sizes at maturity than the migrating clone. The non-migrating clone responded to the fish chemical when it was exposed to it every 24 or 48 h, whereas the migrating clone only responded to the exudate if exposed to it every 24 h.     

Solving the puzzle of metastasis: the evolution of cell migration in neoplasms

Background

Metastasis represents one of the most clinically important transitions in neoplastic progression. The evolution of metastasis is a puzzle because a metastatic clone is at a disadvantage in competition for space and resources with non-metastatic clones in the primary tumor. Metastatic clones waste some of their reproductive potential on emigrating cells with little chance of establishing metastases. We suggest that resource heterogeneity within primary tumors selects for cell migration, and that cell emigration is a by-product of that selection.

Methods and Findings

We developed an agent-based model to simulate the evolution of neoplastic cell migration. We simulated the essential dynamics of neoangiogenesis and blood vessel occlusion that lead to resource heterogeneity in neoplasms. We observed the probability and speed of cell migration that evolves with changes in parameters that control the degree of spatial and temporal resource heterogeneity. Across a broad range of realistic parameter values, increasing degrees of spatial and temporal heterogeneity select for the evolution of increased cell migration and emigration.

Conclusions

We showed that variability in resources within a neoplasm (e.g. oxygen and nutrients provided by angiogenesis) is sufficient to select for cells with high motility. These cells are also more likely to emigrate from the tumor, which is the first step in metastasis and the key to the puzzle of metastasis. Thus, we have identified a novel potential solution to the puzzle of metastasis.     

A low-copy-number Sorghum DNA sequence that detects hypervariable EcoRV fragments

A sorghum genomic DNA clone that hybridized on Southern blots in simple but different patterns to fragments produced by digestion of DNA from the parents of an F₂ mapping population was hybridized to EcoRV-digested DNA from 53 accessions. Forty-six different fragment patterns were observed, each comprised of from one to ten bands. Much less variability was detected in EcoRI than EcoRV digests of a selected subset of the accessions. Base-sequence analysis of the clone did not reveal a functional identity for the sequence and the clone does not contain repeated sequences often associated with hypervariable loci. Clones such as this will be especially useful in evaluating germplasm diversity and in identifying the potential parentage of hybrids.     

Genetic variation in the effect of a facultative symbiont on host-plant use by pea aphids

Ecological specialisation on different host plants occurs frequently among phytophagous insects and is normally assumed to have a genetic basis. However, insects often carry microbial symbionts, which may play a role in the evolution of specialisation. The bacterium Regiella insecticola is a facultative symbiont of pea aphids (Acyrthosiphon pisum) where it is found most frequently in aphid clones feeding on Trifolium giving rise to the hypothesis that it may improve aphid performance on this plant. A study in which R. insecticola was eliminated from a single naturally infected aphid clone supported the hypothesis, but a second involving two aphid clones did not find the same effect. We created a series of new pea aphid–R. insecticola associations by injecting different strains of bacteria into five aphid clones uninfected by symbionts. For all aphid clones, the bacteria decreased the rate at which aphids accepted Vicia faba as a food plant and reduced performance on this plant. Their effect on aphids given Trifolium pratense was more complex: R. insecticola negatively affected acceptance by all aphid clones, had no effect on the performance of four aphid clones, but increased performance of a fifth, thus demonstrating genetic variation in the effect of R. insecticola on pea aphid host use. We discuss how these results may explain the distribution and frequency of this symbiont across different aphid populations. Julia Ferrari and Claire L. Scarborough contributed equally to the work.     

Modeling the connection between primary and metastatic tumors

We put forward a model for cancer metastasis as a migration phenomenon between tumor cell populations coexisting and evolving in two different habitats. One of them is a primary tumor and the other one is a secondary or metastatic tumor. The evolution of the different cell phenotype populations in each habitat is described by means of a simple quasispecies model allowing for a cascade of mutations between the different phenotypes in each habitat. The cell migration event is supposed to be unidirectional and take place continuously in time. The possible clinical outcomes of the model depending on the parameter space are analyzed and the effect of the resection of the primary tumor is studied.     

Isolation and characterization of pollen-specific maize genes with sequence homology to ragweed allergens and pectate lyases

A cDNA clone (Zm58.1) was isolated by differential screening from a cDNA library made to mature Zea mays pollen, and shown to be pollen-specific by RNA blot analysis. When this partial-length clone was used to probe a genomic library, a similar but distinct pollen-specific genomic clone (68% sequence identity) was isolated (Zm58.2). The putative proteins coded for by these two clones show sequence homology to several flower-expressed gene products from various plant species, including known pollen allergens from short ragweed (Ambrosia artemisiifolia), and to pectate lyases from the plant pathogenic bacteria Erwinia spp. The two genes map to different chromosomes.     

The 5S RNA genes inPinus radiata and the spacer region as a probe for relationships betweenPinus species

The 5S RNA genes inPinus radiata occur in two size classes of about 850 and 525 bp in length. Representatives from both the long and short size classes were cloned and sequenced. The primary difference in the two size classes was shown to be a 330 bp insertion in the spacer region of the long units. Within an individual breeding clone ofP. radiata there was some sequence heterogeneity between representatives of the short class. The 5S RNA genes in thirty pine species were characterised using either a clone of the short size class or a subclone of the 330 bp insertion characterizing the long size class. Eleven species of subg.Strobus were examined and all lacked the long type of unit of radiata pine. The New World species of subg.Pinus all had both short and long units whereas the Old World species had long units. The implications of these results for the evolution of the 5S DNA sequences and the phylogenetic relationships withinPinus are discussed.     

Effort to reconstruct past population history in the fern <Emphasis Type="Italic">Blechnum spicant</Emphasis>

Our aim was to evaluate the potential of existing herbarium collections in reconstructing the past population history of the rediscovered clone of the regionally extinct Blechnum spicant by comparing it with herbarium material from previous sites of occurrences in Finland and elsewhere in northern Europe, as well as to reveal the genetic and geographic relationship among the samples. We detected a total of nine polymorphic sites within the three sequenced regions, two SCAR markers developed now for B. spicant and the chloroplast trnL-trnF spacer, totalling 763 bp. Despite low variability, the phylogeographic analysis revealed the presence of some geographic pattern among the samples, of which all except one represented herbarium samples of up to 100 years of age. The rediscovered clone of B. spicant proved to represent the prevalent genotype occurring in northern Europe. The studied sequences were neutral in terms of evolution. It is apparent that existing herbarium collections are useful resources for a range of evolutionary and population studies.     

Ribulose-1, 5-bisphosphate carboxylase activity,chlorophyll and protein concentrations in differentPopulus clones

Ribulose-1,5-bisphosphate carboxylase activity (RuBPC), chlorophyll (chl) and protein (prot) concentrations and chlorophyll/protein (chl/prot) ratios were determined in five differentPopulus clones together with their maximal net CO₂ uptake rates (P_max). A classic reference clone (Populus ×euramericana “Robusta” (Dode) Guinier) was compared with four recently selected euramerican and interamerican crossings. Chl/prot ratio and RuBPC activity varied among the different clones, while chl a/chlb ratio showed only a very low coefficient of variation (1.7%) for the five clones. Poplar clone “Robusta” could be distinguished from the recent faster growing clones based on the different biochemical characteristics. A significant correlation was found between both total chl concentration and chl/prot ratio with P_max for the five clones.     

Cloning of a urochordate cDNA featuring mammalian short consensus repeats (SCR) of complement-control protein superfamily

Mammalian tumor necrosis factor (TNF)-α degenerate polymerase chain reaction (PCR) primers were used to amplify a probe from Botryllus schlosseri (colonial ascidian) allogeneic rejection-cDNA library. A PCR product (269 bp) was cloned and sequenced encoding an open reading frame (ORF) of 89 amino acids (aa). This clone, which revealed no similarity to TNF-α, but a substantial similarity to mammalian proteins featuring short consensus repeats (SCRs) of the complement control superfamily, was used to probe the rejection-cDNA library. Two partial cDNA clones were isolated and sequenced (Bs. 1, 846 bp; Bs.2, 712 bp). The longest ORF in clone Bs. 1 (which lacks the 5' end of the cDNA) predicts a protein of 251 aa, which differs from Bs.2 at six nucleotides and four aa. We compare the as similarity (up to 50.5%) of Bs.l with the SCR-region of mammalian complement factor H, apolipoprotein H, selectins, and complement receptors type 1 and type 2. A somatomedin B-like domain at the C-terminus of Bs. 1 deduced protein was also recorded. We propose that this mosaic and polymorphic botryllid sequence, featuring mammalian-like SCRs, might be an ancestral molecule in the evolution of the chordate's complement-control protein superfamily.     

Cloning of the cDNA and genomic clones for glutathione synthetase fromArabidopsis thaliana and complementation of agsh2 mutant in fission yeast

Glutathione is essential for protecting plants from a range of environmental stresses, including heavy metals where it acts as a precursor for the synthesis of phytochelatins. A 1658 bp cDNA clone for glutathione synthetase (gsh2) was isolated fromArabidopsis thaliana plants that were actively synthesizing glutathione upon exposure to cadmium. The sequence of the clone revealed a protein with an estimated molecular mass of 53858 Da that was very similar to the protein from higher eukaryotes, was less similar to the gene from the fission yeast,Schizosaccharomyces pombe, and shared only a small region of similarity with theEscherichia coli protein. A 4.3 kbSstI fragment containing the genomic clone for glutathione synthetase was also isolated and sequenced. A comparison of the cDNA and genomic sequences revealed that the gene was composed of twelve exons.When theArabidopsis cDNA cloned in a special shuttle vector was expressed in aS. pombe mutant deficient in glutathione synthetase activity, the plant cDNA was able to complement the yeast mutation. Glutathione synthetase activity was measurable in wild-type yeast cells, below detectable levels in thegsh2 ^- mutant, and restored to substantial levels by the expression of theArabidopsis cDNA. TheS. pombe mutant expressing the plant cDNA had near wild type levels of total cellular thiols,¹⁰⁹Cd²⁺ binding activity, and cadmium resistance. Since theArabidopsis cDNA was under control of a thiamine-repressible promoter, growth of the transformed yeast on thiamine-free medium increased expression of the cDNA resulting in increases in cadmium resistance.     

Rapid Phenotypic and Genomic Change in Response to Therapeutic Pressure in Prostate Cancer Inferred by High Content Analysis of Single Circulating Tumor Cells

Timely characterization of a cancer''s evolution is required to predict treatment efficacy and to detect resistance early. High content analysis of single Circulating Tumor Cells (CTCs) enables sequential characterization of genotypic, morphometric and protein expression alterations in real time over the course of cancer treatment. This concept was investigated in a patient with castrate-resistant prostate cancer progressing through both chemotherapy and targeted therapy. In this case study, we integrate across four timepoints 41 genome-wide copy number variation (CNV) profiles plus morphometric parameters and androgen receptor (AR) protein levels. Remarkably, little change was observed in response to standard chemotherapy, evidenced by the fact that a unique clone (A), exhibiting highly rearranged CNV profiles and AR+ phenotype was found circulating before and after treatment. However, clinical response and subsequent progression after targeted therapy was associated with the drastic depletion of clone A, followed by the sequential emergence of two distinct CTC sub-populations that differed in both AR genotype and expression phenotype. While AR- cells with flat or pseudo-diploid CNV profiles (clone B) were identified at the time of response, a new tumor lineage of AR+ cells (clone C) with CNV altered profiles was detected during relapse. We showed that clone C, despite phylogenetically related to clone A, possessed a unique set of somatic CNV alterations, including MYC amplification, an event linked to hormone escape. Interesting, we showed that both clones acquired AR gene amplification by deploying different evolutionary paths. Overall, these data demonstrate the timeframe of tumor evolution in response to therapy and provide a framework for the multi-scale analysis of fluid biopsies to quantify and monitor disease evolution in individual patients.     

Induction of endogenous xenotropic retrovirus expression by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate

Summary The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined for its ability to induce endogenous retrovirus from a high-passage clone of Kirsten sarcoma virus-transformed Balb/c (K-Balb) mouse cells. TPA activated virus in a concentration-dependent manner (0.0016 to 4.0 μM). Exposure to 1mM actinomycin D inhibited virus induction, suggesting that cellular RNA synthesis is required de novo by this inducer. A broad-spectrum neutralizing antibody to murine type C virus, gp70, was shown to neutralize the infectivity of the induced virus. The activated virus had the host range of the xenotropic Balb virus:2, and after removal of the inducer, the activated state decayed rapidly. TPA stimulated DNA, RNA, and protein synthesis in K-Balb cells, indicating that the mechanism of inducation may be different from that of previously identified virus inducers. The effects observed using the well-defined K-Balb system offer an opportunity to study the modulation of retrovirus gene expression by TPA. This work was conducted while the authors were with the Biological Carcinogenesis Program, Frederick Cancer Research Facility, Frederick, MD 21701, and was supported under Contract NO1-CO-75380 with the National Cancer Institute, National Institutes of Health, Bethesda, MD 20205.     

Phylogeny and biogeography in the evolution of migration: shorebirds of the Charadrius complex

Aim We explore whether molecular phylogeny and biogeography can complement evolutionary ecology in developing a method to address a long-standing issue in the evolution of migration: have migrations between breeding and non-breeding grounds, which may be on different continents, evolved through origins in the breeding grounds with successive shifts of the non-breeding distribution or vice versa? Methods To accommodate the biology of migration, we treated breeding and non-breeding distributions as characters to be mapped onto a phylogeny derived from mitochondrial DNA sequence data and so examined the ancestral home issue as a study in the direction of character evolution. Results Our main findings from applying this approach to a subset of the Charadrius complex of shorebirds (Aves: Charadriinae) are that a case can be made for shifts of breeding distributions having occurred in the ancestries of C. alexandrinus and C. veredus as those species evolved their present migration patterns. Our results also argue for a southern hemisphere origin (specifically South America) for the Charadrius complex as a whole. A South American origin implies other shifts in breeding distributions having occurred in the evolution of the species C. semipalmatus and C. vociferus. On applying the methods we developed for dealing with phylogenetic uncertainty, these results are reinforced and the merit of testing them further is suggested. Conclusions By way of a new approach to the evolution of migration, our study adds to a consensus emerging from the evolutionary ecology of migrant birds, arguing that shifts of breeding distributions are commonly, though not necessarily exclusively, involved in the evolution of migration.     

Identification and initial utilization of a portion of the smaller plasmid of Anabaena variabilis ATCC 29413 capable of replication in Anabaena sp. strain M-131

Summary Anabaena variabilis ATCC 29413 contains two cryptic plasmids. Clones of the smaller (41 kb) plasmid, designated pRDS1, in cosmid vectors were used to construct a physical map. A clone bank of pRDS1 constructed by ligating fragments from aXhoII digest of a pRDS1 cosmid clone into a mobilizable plasmid was used to locate an origin of replication of pRDS1. Because we were unable to cureA. variabilis of pRDS1, the clone bank was transferred by conjugation to another strain ofAnabaena sp., strain M-131. A 5.3 kb fragment of pRDS1 contained all of the sequences necessary for replication inAnabaena sp. strain M-131 as judged by the ability to rescue the hybrid vector from exconjugants in unchanged form after many generations. Hybrid plasmids derived from pRDS1, one bearing genes for luciferase, were also transferred by conjugation toA. variabilis, where they appeared to recombine with pRDS1.     

Reproductive effort in clonal plants: constant allocation ratios among ramets?

Summary Armstrong (1982, 1983) predicted that all ramets within a clone should have the same ratio of biomass allocation to sexual reproduction versus vegetative growth. He presented data (1984) that he interpreted as showing that Solidago altissima ramets in a clone do have the predicted constant allocation ratio. Reanalysis of his methods shows that this conclusion was an artifact of his analysis. A simulation using random numbers and Armstrong's analysis showed the same pattern as his data. Data from S. altissima ramets of a single clone grown in a greenhouse experiment, using a different analysis, illustrated that the allocation ratios within a clone can be highly variable.     

Genetic and physical maps around the sex-determining <Emphasis Type="Italic">M</Emphasis>-locus of the dioecious plant asparagus

Asparagus officinalis L. is a dioecious plant. A region called the M-locus located on a pair of homomorphic sex chromosomes controls the sexual dimorphism in asparagus. The aim of this work was to clone the region determining sex in asparagus from its position in the genome. The structure of the region encompassing M should be investigated and compared to the sex-determining regions in other dioecious model species. To establish an improved basis for physical mapping, a high-resolution genetic map was enriched with AFLP markers closely linked to the target locus by carrying out a bulked segregant analysis. By screening a BAC library with AFLP- and STS-markers followed by chromosome walking, a physical map with eight contigs could be established. However, the gaps between the contigs could not be closed due to a plethora of repetitive elements. Surprisingly, two of the contigs on one side of the M-locus did not overlap although they have been established with two markers, which mapped in a distance as low as 0.25 cM flanking the sex locus. Thus, the clustering of the markers indicates a reduced recombination frequency within the M-region. On the opposite side of the M-locus, a contig was mapped in a distance of 0.38 cM. Four closely linked BAC clones were partially sequenced and 64 putative ORFs were identified. Interestingly, only 25% of the ORFs showed sequence similarity to known proteins and ESTs. In addition, an accumulation of repetitive sequences and a low gene density was revealed in the sex-determining region of asparagus. Molecular cytogenetic and sequence analysis of BACs flanking the M-locus indicate that the BACs contain highly repetitive sequences that localize to centromeric and pericentromeric locations on all asparagus chromosomes, which hindered the localization of the M-locus to the single pair of sex chromosomes. We speculate that dioecious Silene, papaya and Asparagus species may represent three stages in the evolution of XX, XY sex determination systems. Given that asparagus still rarely produces hermaphroditic flowers and has homomorphic sex chromosomes, this species may be an ideal system to further investigates early sex chromosome evolution and the origins of dioecy.