A mitogenomic perspective on the ancient, rapid radiation in the Galliformes with an emphasis on the Phasianidae

Background: The Galliformes is a well-known and widely distributed Order in Aves. The phylogenetic relationships of galliform birds, especially the turkeys, grouse, chickens, quails, and pheasants, have been studied intensively, likely because of their close association with humans. Despite extensive studies, convergent morphological evolution and rapid radiation have resulted in conflicting hypotheses of phylogenetic relationships. Many internal nodes have remained ambiguous. Results: We analyzed the complete mitochondrial (mt) genomes from 34 galliform species, including 14 new mt genomes and 20 published mt genomes, and obtained a single, robust tree. Most of the internal branches were relatively short and the terminal branches long suggesting an ancient, rapid radiation. The Megapodiidae formed the sister group to all other galliforms, followed in sequence by the Cracidae, Odontophoridae and Numididae. The remaining clade included the Phasianidae, Tetraonidae and Meleagrididae. The genus Arborophila was the sister group of the remaining taxa followed by Polyplectron. This was followed by two major clades: ((((Gallus, Bambusicola) Francolinus) (Coturnix, Alectoris)) Pavo) and (((((((Chrysolophus, Phasianus) Lophura) Syrmaticus) Perdix) Pucrasia) (Meleagris, Bonasa)) ((Lophophorus, Tetraophasis) Tragopan))). Conclusions: The traditional hypothesis of monophyletic lineages of pheasants, partridges, peafowls and tragopans was not supported in this study. Mitogenomic analyses recovered robust phylogenetic relationships and suggested that the Galliformes formed a model group for the study of morphological and behavioral evolution.

Genetic relationship of Chinese and Japanese gamecocks revealed by mtDNA sequence variation

Cockfighting has a very long history dating back to as early as 2500 years ago in China. Cockfighting was intertwined with human cultural traditions, helped disperse chickens across the world, and influenced the subsequent breed selection. Therefore, trac

藏鸡和姬鼠属(Apodemus )系统进化中的比较基因组学研究

在过去的十几年时间里，利用不同的分子标记促进了分子生态学和群 体遗传学的较大发展，其中线粒体DNA 和微卫星DNA 分析在分子生态学 研究中得到了最为广泛的应用，多核基因分子标记也越来越受到人们的关 注。本文采用了比较基因组学方法来研究了藏鸡(Tibetan Chickens)和姬鼠 属(Apodemus)的系统进化，对藏鸡的研究主要是为了解决藏鸡的系统进化 地位及其线粒体基因组的进化特征；对姬鼠属的研究目的在于发展建立一 种崭新的研究野生动物系统进化和生物地理的比较基因组学手段。主要结 果描述如下： (1)藏鸡(Tibetan Chickens)线粒体全基因组序列的测序和分析 通过利用PCR 扩增，测序，拼接，获得藏鸡线粒体全基因组序列并进 行数据分析处理。藏鸡线粒体全基因组序列全长16783bp，共有13 个蛋白 质编码基因、2 个rRNA 基因、22 个tRNA 基因和1 个D-loop 区。模拟电 子酶切结果显示，藏鸡DraI 酶的酶切结果和其他家鸡及红原鸡的酶切结果 都相同。基于D-loop 区全序列和13 个蛋白质编码基因序列，采用N-J 算 法与原鸡属4 个种，3 个亚种和3 个家鸡品系构建系统进化树：初步确定 藏鸡起源于红原鸡，与家鸡中的来航鸡、白洛克鸡亲缘关系最近，但是藏 鸡的进化与来航鸡、白洛克鸡这两个家鸡品系又显得相对独立。推测可能 原因是藏鸡的祖先在进入高原以后处于相对封闭的环境，从而形成了独特 群体遗传特性。 (2)姬鼠属(Apodemus)系统进化中的比较基因组学研究 本文中我们利用比较基因组学的研究方法寻找Exon-Primer-Intron- Crossing(EPIC)座位，并在中国四川省姬鼠属3 个种18 个个体中进行检验。 其方法是：通过比较人和小家鼠基因组，选择其中的外显子高度保守的单 拷贝基因，然后在500-1500bp 长度内含子的两端利用外显子序列设计了引 物，再进行PCR 扩增和克隆分析。通过PCR 扩增，我们在102 对引物选 择了6 对引物在18 个姬鼠属个体中进行PCR 产物克隆和测序。通过和 Cyt-b 相比较，在6 个座位当中，有5 个座位构建的系统进化树和Cyt-b 构建的系统进化树的拓扑结构高度一致，其中4 个座位可以很好的区分地 理不同的种群。通过计算核酸多样性，6 个座位的得到的结果都很接近， 说明6 个座位的突变率没有太大的差别。由此可见，我们利用比较基因组学的方法寻找EPIC 座位用于系统发育和群体遗传学的研究是可行的，通 过利用模式物种的基因组信息来研究野生非模式物种的系统发育和群体 遗传学将会提供前所未有的数据量和分辨率。

Preparation and mass spectrometric study of egg yolk antibody (IgY) against rabies virus

Rabies virus was used as the antigen to immunize laying chickens. Anti-rabies virus immunoglobulin Y(IgY) was isolated from yolks of the eggs laid by these chickens using a two-step salt precipitation and one-step gel filtration protocol. The purified IgY was reduced with dithiothreitol, and heavy chains (HC) and light chains (LC) were obtained. In addition, the purified IgY was digested with pepsin and the fragment with specific antigen binding properties (Fab) was produced. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOFMS), the average molecular weights of IgY, HC, LC, and Fab were determined as 167 250, 65 105, 18 660, and 45,359 Da, respectively. IgY has two structural differences compared with mammalian IgGs. First, the molecular weight of the heavy chain of IgY is larger than that of its mammalian counterpart, while the molecular weight of the light chain of IgY is smaller. Second, upon pepsin digestion, anti-rabies virus IgY is degraded into Feb, in contrast to mammalian IgG, which has been reported to be degraded into F(ab')(2) under the same conditions. Copyright (C) 2001 John Wiley & Sons, Ltd.

Genetic evaluation of a proposed introduction: the case of the greater prairie chicken and the extinct heath hen.

Population introduction is an important tool for ecosystem restoration. However, before introductions should be conducted, it is important to evaluate the genetic, phenotypic and ecological suitability of possible replacement populations. Careful genetic analysis is particularly important if it is suspected that the extirpated population was unique or genetically divergent. On the island of Martha's Vineyard, Massachusetts, the introduction of greater prairie chickens (Tympanuchus cupido pinnatus) to replace the extinct heath hen (T. cupido cupido) is being considered as part of an ecosystem restoration project. Martha's Vineyard was home to the last remaining heath hen population until its extinction in 1932. We conducted this study to aid in determining the suitability of greater prairie chickens as a possible replacement for the heath hen. We examined mitochondrial control region sequences from extant populations of all prairie grouse species (Tympanuchus) and from museum skin heath hen specimens. Our data suggest that the Martha's Vineyard heath hen population represents a divergent mitochondrial lineage. This result is attributable either to a long period of geographical isolation from other prairie grouse populations or to a population bottleneck resulting from human disturbance. The mtDNA diagnosability of the heath hen contrasts with the network of mtDNA haplotypes of other prairie grouse (T. cupido attwateri, T. pallidicinctus and T. phasianellus), which do not form distinguishable mtDNA groupings. Our findings suggest that the Martha's Vineyard heath hen was more genetically isolated than are current populations of prairie grouse and place the emphasis for future research on examining prairie grouse adaptations to different habitat types to assess ecological exchangeability between heath hens and greater prairie chickens.

Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.

Guanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.

Dopamine receptors in a songbird brain.

Dopamine is a key neuromodulatory transmitter in the brain. It acts through dopamine receptors to affect changes in neural activity, gene expression, and behavior. In songbirds, dopamine is released into the striatal song nucleus Area X, and the levels depend on social contexts of undirected and directed singing. This differential release is associated with differential expression of activity-dependent genes, such as egr1 (avian zenk), which in mammalian brain are modulated by dopamine receptors. Here we cloned from zebra finch brain cDNAs of all avian dopamine receptors: the D1 (D1A, D1B, D1D) and D2 (D2, D3, D4) families. Comparative sequence analyses of predicted proteins revealed expected phylogenetic relationships, in which the D1 family exists as single exon and the D2 family exists as spliced exon genes. In both zebra finch and chicken, the D1A, D1B, and D2 receptors were highly expressed in the striatum, the D1D and D3 throughout the pallium and within the mesopallium, respectively, and the D4 mainly in the cerebellum. Furthermore, within the zebra finch, all receptors, except for D4, showed differential expression in song nuclei relative to the surrounding regions and developmentally regulated expression that decreased for most receptors during the sensory acquisition and sensorimotor phases of song learning. Within Area X, half of the cells expressed both D1A and D2 receptors, and a higher proportion of the D1A-only-containing neurons expressed egr1 during undirected but not during directed singing. Our findings are consistent with hypotheses that dopamine receptors may be involved in song development and social context-dependent behaviors.

Reconstruction of gross avian genome structure, organization and evolution suggests that the chicken lineage most closely resembles the dinosaur avian ancestor.

BACKGROUND: The availability of multiple avian genome sequence assemblies greatly improves our ability to define overall genome organization and reconstruct evolutionary changes. In birds, this has previously been impeded by a near intractable karyotype and relied almost exclusively on comparative molecular cytogenetics of only the largest chromosomes. Here, novel whole genome sequence information from 21 avian genome sequences (most newly assembled) made available on an interactive browser (Evolution Highway) was analyzed. RESULTS: Focusing on the six best-assembled genomes allowed us to assemble a putative karyotype of the dinosaur ancestor for each chromosome. Reconstructing evolutionary events that led to each species' genome organization, we determined that the fastest rate of change occurred in the zebra finch and budgerigar, consistent with rapid speciation events in the Passeriformes and Psittaciformes. Intra- and interchromosomal changes were explained most parsimoniously by a series of inversions and translocations respectively, with breakpoint reuse being commonplace. Analyzing chicken and zebra finch, we found little evidence to support the hypothesis of an association of evolutionary breakpoint regions with recombination hotspots but some evidence to support the hypothesis that microchromosomes largely represent conserved blocks of synteny in the majority of the 21 species analyzed. All but one species showed the expected number of microchromosomal rearrangements predicted by the haploid chromosome count. Ostrich, however, appeared to retain an overall karyotype structure of 2n=80 despite undergoing a large number (26) of hitherto un-described interchromosomal changes. CONCLUSIONS: Results suggest that mechanisms exist to preserve a static overall avian karyotype/genomic structure, including the microchromosomes, with widespread interchromosomal change occurring rarely (e.g., in ostrich and budgerigar lineages). Of the species analyzed, the chicken lineage appeared to have undergone the fewest changes compared to the dinosaur ancestor.

A whole-cell and single-channel study of the voltage-dependent outward potassium current in avian hepatocytes.

Voltage-dependent membrane currents were studied in dissociated hepatocytes from chick, using the patch-clamp technique. All cells had voltage-dependent outward K+ currents; in 10% of the cells, a fast, transient, tetrodotoxin-sensitive Na+ current was identified. None of the cells had voltage-dependent inward Ca2+ currents. The K+ current activated at a membrane potential of about -10 mV, had a sigmoidal time course, and did not inactivate in 500 ms. The maximum outward conductance was 6.6 +/- 2.4 nS in 18 cells. The reversal potential, estimated from tail current measurements, shifted by 50 mV per 10-fold increase in the external K+ concentration. The current traces were fitted by n2 kinetics with voltage-dependent time constants. Omitting Ca2+ from the external bath or buffering the internal Ca2+ with EGTA did not alter the outward current, which shows that Ca2+-activated K+ currents were not present. 1-5 mM 4-aminopyridine, 0.5-2 mM BaCl2, and 0.1-1 mM CdCl2 reversibly inhibited the current. The block caused by Ba was voltage dependent. Single-channel currents were recorded in cell-attached and outside-out patches. The mean unitary conductance was 7 pS, and the channels displayed bursting kinetics. Thus, avian hepatocytes have a single type of K+ channel belonging to the delayed rectifier class of K+ channels.

‘Close as a kiss’: Gyn/Affection in Margaret Atwood’s The Penelopiad

Margaret Atwood’s novella The Penelopiad (2005) seemingly celebrates Penelope’s agency in opposition to Homer’s myth in The Odyssey. However, the twelve murdered maids steal the book to suggest the possibility of what Janice Raymond calls gyn/affection, a female bonding based on the logic of emotion that, in Atwood’s revision, verges on Kristevan abjection, the sinister and the fantastic, and serves a cathartic effect not only in the maids but also in the reader. This essay aims to question the generally accepted empowerment of Atwood’s Penelope and celebrates the murdered maids as the locus of emotion, where marginal aspects of gender and class merge to weave a powerful metaphorical tapestry of popular and traditionally feminized literary genres that, in plunging into and embracing the semiotic realm, ultimately solidify into an eclectic but compact alternative tradition of women’s writing and myth-making.

Rapid screening for monensin residues in poultry plasma by a dry reagent dissociation enhanced lanthanide fluoroimmunoassay.

Monensin, a carboxylic acid ionophore, is commonly fed to poultry to control coccidiosis. A method for rapid analysis of unextracted poultry plasma samples has been developed based on a novel immunoassay format: one-step all-in-one dry reagent time resolved fluorimetry. All assay specific components were pre-dried onto microtitration plate wells. Only addition of the serum sample diluted in assay buffer was required to perform analysis. Results were available one hour after sample addition. The limit of detection (mean + 3s) of the assay calculated from the analysis of 23 known negative samples was 14.2 ng ml(-1). Intra- and inter-assay RSD were determined as 15.2 and 7.4%, respectively, using a plasma sample fortified with 50 ng ml(-1) monensin. Eight broiler chickens were fed monensin at a dose rate of 120 mg kg(-1) feed for one week, blood sampled then slaughtered without drug withdrawal. Plasma monensin concentrations, as determined by the fluoroimmunoassay ranged from 101-297 ng ml(-1). This compared with monensin liver concentrations, determined by LC-MS, which ranged fi om 13-41 ng g(-1). The fluoroimmunoassay described is extremely user friendly, gives particularly rapid results and is suitable for the detection and quantification of plasma monensin residues. Data from medicated poultry suggest that analysis of plasma may be useful in predicting the extent of monensin liver residues.

The effect of glycerol inclusion on broiler performance and nutrient digestibility

1. Crude glycerol from biodiesel production was offered ad libitum to broiler chickens in a 21-d feeding and digestibility trial. The study was designed as a 3*2+1 factorial design with 3 concentrations (33, 67, 100 g/kg) of glycerol from 2 sources, A and B (PRS Environmental Ltd and John Thompson and Sons Ltd) and a control diet. The diets were formulated to contain apparent metabolisable energy (AME) of 12.95 MJ/kg (assuming 14.6 MJ/kg for glycerol).

Nitrofurazone accumulates in avian eyes - a replacement for semicarbazide as a marker of abuse

Intact nitrofurazone is present in whole eyes of chickens fed varying levels of this banned antibiotic and may therefore be used as an alternative to the controversial marker residue, semicarbazide, to monitor for abuse of this drug in primary production.

Effects of orally administered Bdellovibrio bacteriovorus on the well-being and Salmonella colonization of young chicks

Bdellovibrio bacteriovorus is a bacterium which preys upon and kills Gram-negative bacteria, including the zoonotic pathogens Escherichia coli and Salmonella. Bdellovibrio has potential as a biocontrol agent, but no reports of it being tested in living animals have been published, and no data on whether Bdellovibrio might spread between animals are available. In this study, we tried to fill this knowledge gap, using B. bacteriovorus HD100 doses in poultry with a normal gut microbiota or predosed with a colonizing Salmonella strain. In both cases, Bdellovibrio was dosed orally along with antacids. After dosing non-Salmonella-infected birds with Bdellovibrio, we measured the health and well-being of the birds and any changes in their gut pathology and culturable microbiota, finding that although a Bdellovibrio dose at 2 days of age altered the overall diversity of the natural gut microbiota in 28-day-old birds, there were no adverse effects on their growth and well-being. Drinking water and fecal matter from the pens in which the birds were housed as groups showed no contamination by Bdellovibrio after dosing. Predatory Bdellovibrio orally administered to birds that had been predosed with a gut-colonizing Salmonella enterica serovar Enteritidis phage type 4 strain (an important zoonotic pathogen) significantly reduced Salmonella numbers in bird gut cecal contents and reduced abnormal cecal morphology, indicating reduced cecal inflammation, compared to the ceca of the untreated controls or a nonpredatory ΔpilA strain, suggesting that these effects were due to predatory action. This work is a first step to applying Bdellovibrio therapeutically for other animal, and possibly human, infections.

Clathrin is spindle-associated but not essential for mitosis

BACKGROUND: Clathrin is a multimeric protein involved in vesicle coat assembly. Recently clathrin distribution was reported to change during the cell cycle and was found to associate with the mitotic spindle. Here we test whether the recruitment of clathrin to the spindle is indicative of a critical functional contribution to mitosis.

METHODOLOGY/PRINCIPAL FINDINGS: Previously a chicken pre-B lymphoma cell line (DKO-R) was developed in which the endogenous clathrin heavy chain alleles were replaced with the human clathrin heavy chain under the control of a tetracycline-regulatable promoter. Receptor-mediated and fluid-phase endocytosis were significantly inhibited in this line following clathrin knockout, and we used this to explore the significance of clathrin heavy chain expression for cell cycle progression. We confirmed using confocal microscopy that clathrin colocalised with tubulin at mitotic spindles. Using a propidium iodide flow cytometric assay we found no statistical difference in the cell cycle distribution of the knockout cells versus the wild-type. Additionally, we showed that the ploidy and the recovery kinetics following cell cycle arrest with nocodazole were unchanged by repressing clathrin heavy chain expression.

CONCLUSIONS/SIGNIFICANCE: We conclude that the association of clathrin with the mitotic spindle and the contribution of clathrin to endocytosis are evolutionarily conserved. However we find that the contribution of clathrin to mitosis is less robust and dependent on cellular context. In other cell-lines silencing RNA has been used by others to knockdown clathrin expression resulting in an increase in the mitotic index of the cells. We show an effect on the G2/M phase population of clathrin knockdown in HEK293 cells but show that repressing clathrin expression in the DKO-R cell-line has no effect on the size of this population. Consequently this work highlights the need for a more detailed molecular understanding of the recruitment and function of clathrin at the spindle, since the localisation but not the impact of clathrin on mitosis appears to be robust in plants, mammalian and chicken B-cells.