Genomic restructuring in the Tasmanian devil facial tumour: chromosome painting and gene mapping provide clues to evolution of a transmissible tumour

Devil facial tumour disease (DFTD) is a fatal, transmissible malignancy that threatens the world's largest marsupial carnivore, the Tasmanian devil, with extinction. First recognised in 1996, DFTD has had a catastrophic effect on wild devil numbers, and intense research efforts to understand and contain the disease have since demonstrated that the tumour is a clonal cell line transmitted by allograft. We used chromosome painting and gene mapping to deconstruct the DFTD karyotype and determine the chromosome and gene rearrangements involved in carcinogenesis. Chromosome painting on three different DFTD tumour strains determined the origins of marker chromosomes and provided a general overview of the rearrangement in DFTD karyotypes. Mapping of 105 BAC clones by fluorescence in situ hybridisation provided a finer level of resolution of genome rearrangements in DFTD strains. Our findings demonstrate that only limited regions of the genome, mainly chromosomes 1 and X, are rearranged in DFTD. Regions rearranged in DFTD are also highly rearranged between different marsupials. Differences between strains are limited, reflecting the unusually stable nature of DFTD. Finally, our detailed maps of both the devil and tumour karyotypes provide a physical framework for future genomic investigations into DFTD.     

Extreme Telomere Length Dimorphism in the Tasmanian Devil and Related Marsupials Suggests Parental Control of Telomere Length

Telomeres, specialised structures that protect chromosome ends, play a critical role in preserving chromosome integrity. Telomere dynamics in the Tasmanian devil (Sarcophilus harrisii) are of particular interest in light of the emergence of devil facial tumour disease (DFTD), a transmissible malignancy that causes rapid mortality and threatens the species with extinction. We used fluorescent in situ hybridisation to investigate telomere length in DFTD cells, in healthy Tasmanian devils and in four closely related marsupial species. Here we report that animals in the Order Dasyuromorphia have chromosomes characterised by striking telomere length dimorphism between homologues. Findings in sex chromosomes suggest that telomere length dimorphism may be regulated by events in the parental germlines. Long telomeres on the Y chromosome imply that telomere lengthening occurs during spermatogenesis, whereas telomere diminution occurs during oogenesis. Although found in several somatic cell tissue types, telomere length dimorphism was not found in DFTD cancer cells, which are characterised by uniformly short telomeres. This is, to our knowledge, the first report of naturally occurring telomere length dimorphism in any species and suggests a novel strategy of telomere length control. Comparative studies in five distantly related marsupials and a monotreme indicate that telomere dimorphism evolved at least 50 million years ago.     

HDL subfraction distribution of paraoxonase-1 and its relevance to enzyme activity and resistance to oxidative stress

The subfraction distribution of HDL-associated peptides has implications for their functions and the impact of pathological modifications to lipoprotein metabolism on these functions. We have analyzed the subfraction distribution of paraoxonase-1 (PON1) and the consequences for enzyme activity and stability. HDL subfractions were defined by the presence (LpA-I,A-II) or absence (LpA-I) of apolipoprotein A-II (apoA-II). PON1 was present in both subfractions, although increased concentrations of HDL were associated with significantly increased PON1 in LpA-I. ApoA-II did not modify the capacity of native human HDL or reconstituted HDL to promote PON1 secretion from cells or to stabilize enzyme activity, nor did apoA-II decrease PON1 activity when added to rabbit serum normally devoid of the apolipoprotein. LpA-I,A-II particles isolated from human serum or reconstituted HDL (LpA-I,A-II) showed a significantly greater capacity than HDL(LpA-I) to stabilize secreted PON1 and purified recombinant PON1 added to such particles. PON1 associated with apoA-II-containing particles showed greater resistance to inactivation arising from oxidation. ApoA-I, apoA-II, and LpA-I,A-II, but not LpA-I, were independent determinants of serum PON1 concentration and activity in multivariate analyses. PON1 is at least equally distributed between LpA-I and LpA-II,A-II HDL particles. This dichotomous distribution has implications for PON1 activity and stability that may impact on the physiological role of the enzyme.     

Platypus globin genes and flanking loci suggest a new insertional model for beta-globin evolution in birds and mammals

Background  

Vertebrate alpha (α)- and beta (β)-globin gene families exemplify the way in which genomes evolve to produce functional complexity. From tandem duplication of a single globin locus, the α- and β-globin clusters expanded, and then were separated onto different chromosomes. The previous finding of a fossil β-globin gene (ω) in the marsupial α-cluster, however, suggested that duplication of the α-β cluster onto two chromosomes, followed by lineage-specific gene loss and duplication, produced paralogous α- and β-globin clusters in birds and mammals. Here we analyse genomic data from an egg-laying monotreme mammal, the platypus (Ornithorhynchus anatinus), to explore haemoglobin evolution at the stem of the mammalian radiation.     

Detection range of songbirds using a stopover site by automated radio‐telemetry

A major uncertainty in automated radio‐telemetry studies of small birds is the detection range of receiving antennas. We compared simultaneous daytime detections (± 30 s) by automated and manual radio‐telemetry to assess detection probability and the proportion of transmissions detected for birds on migratory stopover as a function of distance, foraging guild (Black‐throated Blue Warblers, Setophaga caerulescens, and Yellow‐rumped Warblers, Dendroica coronata coronata, represented mid‐canopy foliage gleaners and White‐throated Sparrows, Zonotrichia albicollis, represented a ground forager), habitat type, meteorological variables, tower antenna number (1–4), and the position of a bird relative to the receiving antenna's bearing (offset angle). Our study was conducted at a migratory stopover site in southern Ontario, Canada. Most detections were in dense to sparse forest, and all individuals were within 1.03 km of the automated receiving station. Daily detection probability was near 100% for both foraging guilds. However, within 30 s before and after a manual radio‐telemetry location was made, detection probability and the proportion of transmissions detected by automated radio‐telemetry declined with distance, was higher for warblers than sparrows, and was lowest for 90° offset angles. Our results suggest that when research goals do not require detections with high temporal frequency, e.g., estimation of departure date or daily departure probability, our study design had an effective detection range of at least 1 km. However, where temporal precision is required, e.g., to investigate movements and changes in activity levels during stopover, detection range was ~300 m for ground‐foraging sparrows and 600 m for mid‐canopy foraging warblers, which is much lower than the presumed detection range of antennas under optimal conditions (15 km). This corresponds to a spatial area of coverage for forest‐dwelling birds of ~0.3–1.1 km². Our results suggest that to optimally configure an automated radio‐telemetry array at the regional scale, investigators should carefully consider detection range and its underlying covariates, including species type, the habitat matrix, and the orientation of antennas relative to preferred habitat.     

Impact of next-generation sequencing error on analysis of barcoded plasmid libraries of known complexity and sequence

Barcoded vectors are promising tools for investigating clonal diversity and dynamics in hematopoietic gene therapy. Analysis of clones marked with barcoded vectors requires accurate identification of potentially large numbers of individually rare barcodes, when the exact number, sequence identity and abundance are unknown. This is an inherently challenging application, and the feasibility of using contemporary next-generation sequencing technologies is unresolved. To explore this potential application empirically, without prior assumptions, we sequenced barcode libraries of known complexity. Libraries containing 1, 10 and 100 Sanger-sequenced barcodes were sequenced using an Illumina platform, with a 100-barcode library also sequenced using a SOLiD platform. Libraries containing 1 and 10 barcodes were distinguished from false barcodes generated by sequencing error by a several log-fold difference in abundance. In 100-barcode libraries, however, expected and false barcodes overlapped and could not be resolved by bioinformatic filtering and clustering strategies. In independent sequencing runs multiple false-positive barcodes appeared to be represented at higher abundance than known barcodes, despite their confirmed absence from the original library. Such errors, which potentially impact barcoding studies in an application-dependent manner, are consistent with the existence of both stochastic and systematic error, the mechanism of which is yet to be fully resolved.