BackgroundCarboxylesterases (CES) perform diverse metabolic roles in mammalian organisms in the detoxification of a broad range of drugs and xenobiotics and may also serve in specific roles in lipid, cholesterol, pheromone and lung surfactant metabolism. Five CES families have been reported in mammals with human CES1 and CES2 the most extensively studied. Here we describe the genetics, expression and phylogeny of CES isozymes in the opossum and report on the sequences and locations of CES1, CES2 and CES6 'like' genes within two gene clusters on chromosome one. We also discuss the likely sequence of gene duplication events generating multiple CES genes during vertebrate evolution.ResultsWe report a cDNA sequence for an opossum CES and present evidence for CES1 and CES2 like genes expressed in opossum liver and intestine and for distinct gene locations of five opossum CES genes,CES1, CES2.1, CES2.2, CES2.3 and CES6, on chromosome 1. Phylogenetic and sequence alignment studies compared the predicted amino acid sequences for opossum CES with those for human, mouse, chicken, frog, salmon and Drosophila CES gene products. Phylogenetic analyses produced congruent phylogenetic trees depicting a rapid early diversification into at least five distinct CES gene family clusters: CES2, CES1, CES7, CES3, and CES6. Molecular divergence estimates based on a Bayesian relaxed clock approach revealed an origin for the five mammalian CES gene families between 328–378 MYA.ConclusionThe deduced amino acid sequence for an opossum cDNA was consistent with its identity as a mammalian CES2 gene product (designated CES2.1). Distinct gene locations for opossum CES1 (1: 446,222,550–446,274,850), three CES2 genes (1: 677,773,395–677,927,030) and a CES6 gene (1: 677,585,520–677,730,419) were observed on chromosome 1. Opossum CES1 and multiple CES2 genes were expressed in liver and intestine. Amino acid sequences for opossum CES1 and three CES2 gene products revealed conserved residues previously reported for human CES1 involved in catalysis, ligand binding, tertiary structure and organelle localization. Phylogenetic studies indicated the gene duplication events which generated ancestral mammalian CES genes predated the common ancestor for marsupial and eutherian mammals, and appear to coincide with the early diversification of tetrapods.

Baboons (genus Papio) are broadly studied in the wild and in captivity. They are widely used as a nonhuman primate model for biomedical studies, and the Southwest National Primate Research Center (SNPRC) at Texas Biomedical Research Institute has maintained a large captive baboon colony for more than 50 yr. Unlike other model organisms, however, the genomic resources for baboons are severely lacking. This has hindered the progress of studies using baboons as a model for basic biology or human disease. Here, we describe a data set of 100 high-coverage whole-genome sequences obtained from the mixed colony of olive (P. anubis) and yellow (P. cynocephalus) baboons housed at the SNPRC. These data provide a comprehensive catalog of common genetic variation in baboons, as well as a fine-scale genetic map. We show how the data can be used to learn about ancestry and admixture and to correct errors in the colony records. Finally, we investigated the consequences of inbreeding within the SNPRC colony and found clear evidence for increased rates of infant mortality and increased homozygosity of putatively deleterious alleles in inbred individuals.

24 25 26 RUNNING TITLE 27 Genomic analysis of a pedigreed baboon colony 28 29 30 ABSTRACT 35 Baboons (genus Papio) are broadly studied in the wild and in captivity. They are widely 36 used as a non-human primate model for biomedical studies, and the Southwest National Primate 37Research Center (SNPRC) at Texas Biomedical Research Institute has maintained a large captive 38 baboon colony for more than 50 years. Unlike other model organisms though, the genomic 39 resources for baboons are severely lacking. This has hindered the progress of studies using 40 baboons as a model for basic biology or human disease. Here, we describe a dataset of 100 high-41coverage whole-genome sequences obtained from the mixed colony of olive (P. anubis) and 42 yellow (P. cynocephalus) baboons housed at the SNPRC. These data provide a comprehensive 43 catalog of common genetic variation in baboons, as well as a fine-scale genetic map. We show 44 how the data can be used to learn about ancestry and admixture, and to correct errors in the 45 colony records. Finally, we investigated the consequences of inbreeding within the SNPRC 46 colony and found clear evidence for increased rates of juvenile mortality and increased 47 homozygosity of putatively deleterious alleles in inbred individuals. 48 49

in laboratory opossums exhibiting a high lipemic response to dietary cholesterol and fat. Am J Physiol Gastrointest Liver Physiol 303: G12-G19, 2012. First published May 3, 2012 doi:10.1152/ajpgi.00415.2011.-Plasma VLDL and LDL cholesterol were markedly elevated (Ͼ40-fold) in high-responding opossums, but moderately elevated (6-fold) in low-responding opossums after they had consumed a high-cholesterol and high-fat diet for 24 wk. In both high-and low-responding opossums, plasma triglycerides were slightly elevated, threefold and twofold, respectively. Dietary challenge also induced fatty livers in high responders, but not in low responders. We studied the lipid composition, histopathological features, and gene expression patterns of the fatty livers. Free cholesterol (2-fold), esterified cholesterol (11-fold), and triglycerides (2-fold) were higher in the livers of high responders than those in low responders, whereas free fatty acid levels were similar. The fatty livers of high responders showed extensive lobular disarray by histology. Inflammatory cells and ballooned hepatocytes were also present, as were perisinusoidal fibrosis and ductular proliferation. In contrast, liver histology was normal in low responders. Hepatic gene expression revealed differences associated with the development of steatohepatitis in high responders. The accumulation of hepatic cholesterol was concomitant with upregulation of the HMGCR gene and downregulation of the CYP27A1, ABCG8, and ABCB4 genes. Genes involved in inflammation (TNF, NFKB1, and COX2) and in oxidative stress (CYBA and NCF1) were upregulated. Upregulation of the growth factor genes (PDGF and TGFB1) and collagen genes (Col1A1, Col3A1, and Col4A1) was consistent with fibrosis. Some of the histological characteristics of the fatty livers of high-responding opossums imitate those in the livers of humans with nonalcoholic steatohepatitis. ABCB4; hypercholesterolemia; Monodelphis domestica; nonalcoholic fatty liver disease; nonalcoholic steatohepatitis THE GRAY SHORT-TAILED OPOSSUM (Monodelphis domestica) has been established as a laboratory animal for biomedical research (39). Inbreeding and selection for high-or low-plasma cholesterol in response to a high-cholesterol and high-fat (HCHF) diet led to the development of partially inbred strains of laboratory opossums that exhibit dramatic differences in plasma cholesterol when they are challenged with the HCHF diet. After an 8-wk challenge, the HCHF diet induces a marked increase (Ͼ10-fold) in VLDL and LDL cholesterol (VϩLDLC) in high responders, but only a slight increase (Ͻ2-fold) in VϩLDLC in low responders. However, plasma VϩLDLC levels of high and low responders do not differ on the basal diet (17, 28).In search of genes that mediate diet-induced hypercholesterolemia in high responders, we observed that the levels of phospholipids and cholesterol in gall bladder bile were significantly lower in high responders relative to those in low responders on the HCHF diet (4). This observation led us to focus on the ABCB4 gen...

We examined the involvement of the cyclin-dependent kinase inhibitor 2A (CDKN2A) locus in the pathogenesis of ultraviolet (UV) radiation-induced melanomas in an opossum (Monodelphis domestica) melanoma model in which suckling young were exposed to UVB to produce melanocytic lesions. Monodelphis CDKN2A and alternated reading frame (ARF) cDNAs were cloned and sequenced, and the expression patterns of these genes were determined by reverse transcription-polymerase chain reaction in normal tissues, 39 primary melanocytic skin lesions, and two tumor-derived cell lines, one nonmetastatic and one metastatic. Primary melanocytic lesions, including hyperplasias, benign melanomas, melanomas metastatic to lymph nodes, and melanomas metastatic to nodes and additional visceral organs, were categorized accordingly as types I-IV. Levels of CDKN2A transcripts were most abundant in type III tumor samples and the metastatic cell line but absent in the nonmetastatic cell line. ARF transcripts were expressed in all tumors and cell lines. A UV-signature mutation was detected with the wild-type allele at the CDKN2A locus in type II and III primary tumor samples and in the nonmetastatic cell line. Interestingly, in the metastatic cell line, only the mutant allele was present and expressed. These data suggest dynamic changes in the expression and/or structure of the CDKN2A and ARF genes represent one molecular defect associated with the etiology of melanoma formation and progression in the Monodelphis model system.

Bile salts, cholesterol and phosphatidylcholine are secreted across the canalicular membrane of hepatocytes into bile by ATP-binding cassette (ABC) transporters. Secretion of bile salts by ABCB11 is essential for bile flow and for absorption of lipids and fat-soluble vitamins. ABCG5 and ABCG8 eliminate excess cholesterol and sterols from the body by secreting them into bile. There are two mechanisms to protect the canalicular membrane from solubilization by bile salts; ABCB4 secretes phosphatidylcholine into bile to form mixed micelles with bile salts, and ATP8B1 maintains the canalicular membrane in a liquid-ordered state. Three different forms of progressive familial intrahepatic cholestasis (PFIC) disorders, PFIC1, PFIC2 and PFIC3, are caused by mutations in ATP8B1, ABCB11 and ABCB4, respectively. Sitosterolemia is caused by mutations in ABCG5 and ABCG8. This article reviews the physiological roles of these canalicular transporters, and the pathophysiological processes and clinical features associated with their mutations.

This article is available online at http://www.jlr.org similar to a natural human diet in being balanced between vegetable and animal sources and including moderate levels of cholesterol. The predominant form of hypercholesterolemia in Western human societies is caused by a response to dietary cholesterol and fat. However, identification of the genes that control response has been elusive in research involving human subjects or conventional animal models.The discovery that some laboratory opossums develop an extreme lipemic response to dietary cholesterol whereas others have a minimal response ( 1 ) provided an opportunity to identify one or more genes that affect diet-induced hypercholesterolemia and to determine mechanisms by which those genes infl uence cholesterol homeostasis. Therefore, we developed a partially inbred strain (ATHH) that develops highly elevated levels (10-to 30-fold) of very low density and low density lipoprotein cholesterol (V+LDLC) when fed a high-cholesterol, high-fat (HCHF) or a highcholesterol, low-fat (HCLF) diet ( 2, 3 ), and two other partially inbred strains (ATHE and ATHL) that only develop slightly elevated levels (<2-fold) of V+LDLC when fed the same cholesterol-enriched diets. The V+LDLC levels of highand low-responding opossums do not differ on a basal diet.Using these strains, we demonstrated differences between high and low responders in cholesterol absorption ( 4 ), cholesterol metabolism ( 5 ), and expression of genes that regulate cholesterol homeostasis ( 6, 7 ). Genetic analyses of plasma lipoprotein cholesterol levels from pedigreed families implicated a recessive allele at a single gene locus as largely responsible for diet-induced hypercholesterolemia in high-responding opossums ( 8 ), but neither the identity of the locus nor of any candidate gene was identifi ed. The laboratory opossum ( Monodelphis domestica ) is an omnivorous mammal. Unlike mice, rabbits, and other small mammals typically used in research on genetic control of plasma lipids, this species has a natural diet that is Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, ␥ -glutamyl transferase; HCHF, high cholesterol and high fat; HCLF, high cholesterol and low fat; HDLC, high density lipoprotein cholesterol; LD, linkage disequilibrium; SNP, single nucleotide polymorphism; TC, total cholesterol; TG, triglyceride; V+LDLC, very low density and low density lipoprotein cholesterol.

support-information-section).