Christina Williams

The genome of the green anole lizard and a comparative analysis with birds and mammals

The evolution of the amniotic egg was one of the great evolutionary innovations in the history of life, freeing vertebrates from an obligatory connection to water and thus permitting the conquest of terrestrial environments. Among amniotes, genome sequences are available for mammals and birds, but not for non-avian reptiles. Here we report the genome sequence of the North American green anole lizard, Anolis carolinensis. We find that A. carolinensis microchromosomes are highly syntenic with chicken microchromosomes, yet do not exhibit the high GC and low repeat content that are characteristic of avian microchromosomes. Also, A. carolinensis mobile elements are very young and diverse—more so than in any other sequenced amniote genome. The GC content of this lizard genome is also unusual in its homogeneity, unlike the regionally variable GC content found in mammals and birds. We describe and assign sequence to the previously unknown A. carolinensis X chromosome. Comparative gene analysis shows that amniote egg proteins have evolved significantly more rapidly than other proteins. An anole phylogeny resolves basal branches to illuminate the history of their repeated adaptive radiations.

Three crocodilian genomes reveal ancestral patterns of evolution among archosaurs

INTRODUCTION Crocodilians and birds are the two extant clades of archosaurs, a group that includes the extinct dinosaurs and pterosaurs. Fossils suggest that living crocodilians (alligators, crocodiles, and gharials) have a most recent common ancestor 80 to 100 million years ago. Extant crocodilians are notable for their distinct morphology, limited intraspecific variation, and slow karyotype evolution. Despite their unique biology and phylogenetic position, little is known about genome evolution within crocodilians. Evolutionary rates of tetrapods inferred from DNA sequences anchored by ultraconserved elements. Evolutionary rates among reptiles vary, with especially low rates among extant crocodilians but high rates among squamates. We have reconstructed the genomes of the common ancestor of birds and of all archosaurs (shown in gray silhouette, although the morphology of these species is uncertain). RATIONALE Genome sequences for the American alligator, saltwater crocodile, and Indian gharial—representatives of all three extant crocodilian families—were obtained to facilitate better understanding of the unique biology of this group and provide a context for studying avian genome evolution. Sequence data from these three crocodilians and birds also allow reconstruction of the ancestral archosaurian genome. RESULTS We sequenced shotgun genomic libraries from each species and used a variety of assembly strategies to obtain draft genomes for these three crocodilians. The assembled scaffold N50 was highest for the alligator (508 kilobases). Using a panel of reptile genome sequences, we generated phylogenies that confirm the sister relationship between crocodiles and gharials, the relationship with birds as members of extant Archosauria, and the outgroup status of turtles relative to birds and crocodilians. We also estimated evolutionary rates along branches of the tetrapod phylogeny using two approaches: ultraconserved element–anchored sequences and fourfold degenerate sites within stringently filtered orthologous gene alignments. Both analyses indicate that the rates of base substitution along the crocodilian and turtle lineages are extremely low. Supporting observations were made for transposable element content and for gene family evolution. Analysis of whole-genome alignments across a panel of reptiles and mammals showed that the rate of accumulation of micro-insertions and microdeletions is proportionally lower in crocodilians, consistent with a single underlying cause of a reduced rate of evolutionary change rather than intrinsic differences in base repair machinery. We hypothesize that this single cause may be a consistently longer generation time over the evolutionary history of Crocodylia. Low heterozygosity was observed in each genome, consistent with previous analyses, including the Chinese alligator. Pairwise sequential Markov chain analysis of regional heterozygosity indicates that during glacial cycles of the Pleistocene, each species suffered reductions in effective population size. The reduction was especially strong for the American alligator, whose current range extends farthest into regions of temperate climates. CONCLUSION We used crocodilian, avian, and outgroup genomes to reconstruct 584 megabases of the archosaurian common ancestor genome and the genomes of key ancestral nodes. The estimated accuracy of the archosaurian genome reconstruction is 91% and is higher for conserved regions such as genes. The reconstructed genome can be improved by adding more crocodilian and avian genome assemblies and may provide a unique window to the genomes of extinct organisms such as dinosaurs and pterosaurs. To provide context for the diversification of archosaurs—the group that includes crocodilians, dinosaurs, and birds—we generated draft genomes of three crocodilians: Alligator mississippiensis (the American alligator), Crocodylus porosus (the saltwater crocodile), and Gavialis gangeticus (the Indian gharial). We observed an exceptionally slow rate of genome evolution within crocodilians at all levels, including nucleotide substitutions, indels, transposable element content and movement, gene family evolution, and chromosomal synteny. When placed within the context of related taxa including birds and turtles, this suggests that the common ancestor of all of these taxa also exhibited slow genome evolution and that the comparatively rapid evolution is derived in birds. The data also provided the opportunity to analyze heterozygosity in crocodilians, which indicates a likely reduction in population size for all three taxa through the Pleistocene. Finally, these data combined with newly published bird genomes allowed us to reconstruct the partial genome of the common ancestor of archosaurs, thereby providing a tool to investigate the genetic starting material of crocodilians, birds, and dinosaurs.

Genome-wide Association Study Identifies Shared Risk Loci Common to Two Malignancies in Golden Retrievers

Dogs, with their breed-determined limited genetic background, are great models of human disease including cancer. Canine B-cell lymphoma and hemangiosarcoma are both malignancies of the hematologic system that are clinically and histologically similar to human B-cell non-Hodgkin lymphoma and angiosarcoma, respectively. Golden retrievers in the US show significantly elevated lifetime risk for both B-cell lymphoma (6%) and hemangiosarcoma (20%). We conducted genome-wide association studies for hemangiosarcoma and B-cell lymphoma, identifying two shared predisposing loci. The two associated loci are located on chromosome 5, and together contribute ~20% of the risk of developing these cancers. Genome-wide p-values for the top SNP of each locus are 4.6×10-7 and 2.7×10-6, respectively. Whole genome resequencing of nine cases and controls followed by genotyping and detailed analysis identified three shared and one B-cell lymphoma specific risk haplotypes within the two loci, but no coding changes were associated with the risk haplotypes. Gene expression analysis of B-cell lymphoma tumors revealed that carrying the risk haplotypes at the first locus is associated with down-regulation of several nearby genes including the proximal gene TRPC6, a transient receptor Ca2+-channel involved in T-cell activation, among other functions. The shared risk haplotype in the second locus overlaps the vesicle transport and release gene STX8. Carrying the shared risk haplotype is associated with gene expression changes of 100 genes enriched for pathways involved in immune cell activation. Thus, the predisposing germ-line mutations in B-cell lymphoma and hemangiosarcoma appear to be regulatory, and affect pathways involved in T-cell mediated immune response in the tumor. This suggests that the interaction between the immune system and malignant cells plays a common role in the tumorigenesis of these relatively different cancers.

Development of a brain metastatic canine prostate cancer cell line

Prostate cancer in men has a high mortality and morbidity due to metastatic disease. The pathobiology of prostate cancer metastasis is not well understood and cell lines and animal models that recapitulate the complex nature of the disease are needed. Therefore, the goal of the study was to establish and characterize a new prostate cancer line derived from a dog with spontaneous prostate cancer.

Definition of the Cattle Killer Cell Ig–like Receptor Gene Family: Comparison with Aurochs and Human Counterparts

Under selection pressure from pathogens, variable NK cell receptors that recognize polymorphic MHC class I evolved convergently in different species of placental mammal. Unexpectedly, diversified killer cell Ig–like receptors (KIRs) are shared by simian primates, including humans, and cattle, but not by other species. Whereas much is known of human KIR genetics and genomics, knowledge of cattle KIR is limited to nine cDNA sequences. To facilitate comparison of the cattle and human KIR gene families, we determined the genomic location, structure, and sequence of two cattle KIR haplotypes and defined KIR sequences of aurochs, the extinct wild ancestor of domestic cattle. Larger than its human counterpart, the cattle KIR locus evolved through successive duplications of a block containing ancestral KIR3DL and KIR3DX genes that existed before placental mammals. Comparison of two cattle KIR haplotypes and aurochs KIR show the KIR are polymorphic and the gene organization and content appear conserved. Of 18 genes, 8 are functional and 10 were inactivated by point mutation. Selective inactivation of KIR3DL and activating receptor genes leaves a functional cohort of one inhibitory KIR3DL, one activating KIR3DX, and six inhibitory KIR3DX. Functional KIR diversity evolved from KIR3DX in cattle and from KIR3DL in simian primates. Although independently evolved, cattle and human KIR gene families share important function-related properties, indicating that cattle KIR are NK cell receptors for cattle MHC class I. Combinations of KIR and MHC class I are the major genetic factors associated with human disease and merit investigation in cattle.

CD40 ligand is necessary and sufficient to support primary diffuse large B-cell lymphoma cells in culture: a tool for in vitro preclinical studies with primary B-cell malignancies

Abstract Established cell lines are utilized extensively to study tumor biology and preclinical therapeutic development. However, they may not accurately recapitulate the heterogeneity of their corresponding primary disease. B-cell tumor cells are especially difficult to maintain under conventional culture conditions, limiting access to samples that faithfully represent this disease for preclinical studies. Here, we used primary canine diffuse large B-cell lymphoma to establish a culture system that reliably supports the growth of these cells. CD40 ligand, either expressed by feeder cells or provided as a soluble two-trimeric form, was sufficient to support primary lymphoma cells in vitro. The tumor cells retained their original phenotype, clonality and known karyotypic abnormalities after extended expansion in culture. Finally, we illustrate the utility of the feeder cell-free culture system for comparable assessment of cytotoxicity using dog and human B-cell malignancies. We conclude that this system has broad applications for in vitro preclinical development for B-cell malignancies.

Intracoronary allogeneic cardiosphere-derived stem cells are safe for use in dogs with dilated cardiomyopathy

Cardiosphere‐derived cells (CDCs) have been shown to reduce scar size and increase viable myocardium in human patients with mild/moderate myocardial infarction. Studies in rodent models suggest that CDC therapy may confer therapeutic benefits in patients with non‐ischaemic dilated cardiomyopathy (DCM). We sought to determine the safety and efficacy of allogeneic CDC in a large animal (canine) model of spontaneous DCM. Canine CDCs (cCDCs) were grown from a donor dog heart. Similar to human CDCs, cCDCs express CD105 and are slightly positive for c‐kit and CD90. Thirty million of allogeneic cCDCs was infused into the coronary vessels of Doberman pinscher dogs with spontaneous DCM. Adverse events were closely monitored, and cardiac functions were measured by echocardiography. No adverse events occurred during and after cell infusion. Histology on dog hearts (after natural death) revealed no sign of immune rejection from the transplanted cells.

Canine prostate cancer cell line (Probasco) produces osteoblastic metastases in vivo

In 2012, over 240,000 men were diagnosed with prostate cancer and over 28,000 died from the disease. Animal models of prostate cancer are vital to understanding its pathogenesis and developing therapeutics. Canine models in particular are useful due to their similarities to late‐stage, castration‐resistant human disease with osteoblastic bone metastases. This study established and characterized a novel canine prostate cancer cell line that will contribute to the understanding of prostate cancer pathogenesis.

A comprehensive genomic history of extinct and living elephants

Significance Elephantids were once among the most widespread megafaunal families. However, only three species of this family exist today. To reconstruct their evolutionary history, we generated 14 genomes from living and extinct elephantids and from the American mastodon. While previous studies examined only simple bifurcating relationships, we found that gene flow between elephantid species was common in the past. Straight-tusked elephants descend from a mixture of three ancestral populations related to the ancestor of African elephants, woolly mammoths, and present-day forest elephants. We detected interbreeding between North American woolly and Columbian mammoths but found no evidence of recent gene flow between forest and savanna elephants, demonstrating that both gene flow and isolation have been central in the evolution of elephantids. Elephantids are the world’s most iconic megafaunal family, yet there is no comprehensive genomic assessment of their relationships. We report a total of 14 genomes, including 2 from the American mastodon, which is an extinct elephantid relative, and 12 spanning all three extant and three extinct elephantid species including an ∼120,000-y-old straight-tusked elephant, a Columbian mammoth, and woolly mammoths. Earlier genetic studies modeled elephantid evolution via simple bifurcating trees, but here we show that interspecies hybridization has been a recurrent feature of elephantid evolution. We found that the genetic makeup of the straight-tusked elephant, previously placed as a sister group to African forest elephants based on lower coverage data, in fact comprises three major components. Most of the straight-tusked elephant’s ancestry derives from a lineage related to the ancestor of African elephants while its remaining ancestry consists of a large contribution from a lineage related to forest elephants and another related to mammoths. Columbian and woolly mammoths also showed evidence of interbreeding, likely following a latitudinal cline across North America. While hybridization events have shaped elephantid history in profound ways, isolation also appears to have played an important role. Our data reveal nearly complete isolation between the ancestors of the African forest and savanna elephants for ∼500,000 y, providing compelling justification for the conservation of forest and savanna elephants as separate species.

Malignant canine mammary tumours: Preliminary genomic insights using oligonucleotide array comparative genomic hybridisation analysis

Neoplastic mammary disease in female dogs represents a major health concern for dog owners and veterinarians, but the genomic basis of the disease is poorly understood. In this study, we performed high resolution oligonucleotide array comparative genomic hybridisation (oaCGH) to assess genome wide DNA copy number changes in 10 malignant canine mammary tumours from seven female dogs, including multiple tumours collected at one time from each of three female dogs. In all but two tumours, genomic imbalances were detected, with losses being more common than gains. Canine chromosomes 9, 22, 26, 27, 34 and X were most frequently affected. Dissimilar oaCGH ratio profiles were observed in multiple tumours from the same dogs, providing preliminary evidence for probable independent pathogenesis. Analysis of adjacent samples of one tumour revealed regional differences in the number of genomic imbalances, suggesting heterogeneity within tumours.