Comparative molecular cytogenetic studies in the order Carnivora: mapping chromosomal rearrangements onto the phylogenetic tree

We have made a set of chromosome-specific painting probes for the American mink by degenerate oligonucleotide primed-PCR (DOP-PCR) amplification of flow-sorted chromosomes. The painting probes were used to delimit homologous chromosomal segments among human, red fox, dog, cat and eight species of the family Mustelidae, including the European mink, steppe and forest polecats, least weasel, mountain weasel, Japanese sable, striped polecat, and badger. Based on the results of chromosome painting and G-banding, comparative maps between these species have been established. The integrated map demonstrates a high level of karyotype conservation among mustelid species. Comparative analysis of the conserved chromosomal segments among mustelids and outgroup species revealed 18 putative ancestral autosomal segments that probably represent the ancestral chromosomes, or chromosome arms, in the karyotype of the most recent ancestor of the family Mustelidae. The proposed 2n = 38 ancestral Mustelidae karyotype appears to have been retained in some modern mustelids, e.g., Martes, Lutra, ktonyx, and Vormela. The derivation of the mustelid karyotypes from the putative ancestral state resulted from centric fusions, fissions, the addition of heterochromatic arms, and occasional pericentric inversions. Our results confirm many of the evolutionary conclusions suggested by other data and strengthen the topology of the carnivore phylogenetic tree through the inclusion of genome-wide chromosome rearrangements. Copyright (C) 2002 S. KargerAG, Basel.

Avian comparative genomics: reciprocal chromosome painting between domestic chicken (Gallus gallus) and the stone curlew (Burhinus oedicnemus, Charadriiformes) - An atypical species with low diploid number

The chicken is the most extensively studied species in birds and thus constitutes an ideal reference for comparative genomics in birds. Comparative cytogenetic studies indicate that the chicken has retained many chromosome characters of the ancestral avia

Defining the orientation of the tandem fusions that occurred during the evolution of Indian muntjac chromosomes by BAC mapping

The Indian muntjac (Muntiacus muntjak vaginalis) has a karyotype of 2n=6 in the female and 7 in the male, the karyotypic evolution of which through extensive tandem fusions and several centric fusions has been well-documented by recent molecular cytogenetic studies. In an attempt to define the fusion orientations of conserved chromosomal segments and the molecular mechanisms underlying the tandem fusions, we have constructed a highly redundant (more than six times of whole genome coverage) bacterial artificial chromosome (BAC) library of Indian muntjac. The BAC library contains 124,800 clones with no chromosome bias and has an average insert DNA size of 120 kb. A total of 223 clones have been mapped by fluorescent in situ hybridization onto the chromosomes of both Indian muntjac and Chinese muntjac and a high-resolution comparative map has been established. Our mapping results demonstrate that all tandem fusions that occurred during the evolution of Indian muntjac karyotype from the acrocentric 2n=70 hypothetical ancestral karyotype are centromere-telomere (head-tail) fusions.

中国翼手目六个类群的比较分子细胞遗传学研究

翼手类(Order Chiroptera)是哺乳动物中最为神秘的类群之一。它们也是哺乳动物中唯一真正具有空中飞翔能力的动物，而且多数物种还具有回声定位功能。翼手目分布十分广泛，除南极洲之外全球几乎任何地方都有分布，其数量约占整个哺乳动物种类的20％。然而，有关该类群的许多系统发育假说仍存有争议，至今其进化历史还不是很清楚，并且形态和分子研究所得出的结论经常不一致。近年来分子细胞遗传学的发展,特别是比较染色体涂色及其相关技术的应用，使人们可以快速、准确地鉴定种间的同源染色体（或同源染色体片段）。根据构建的染色体同源图谱及外群比较可确定一些细胞遗传学特征，并将其用于系统发育研究。虽然比较染色体涂色技术已广泛应用在哺乳动物的核型进化和系统发育研究中，但是在翼手目中却相当滞后。到目前为止，仅有少数蝙蝠物种有染色体涂色数据。 本研究分别利用三叶小蹄蝠（蹄蝠科）、大鼠耳蝠（蝙蝠科）及人的染色体特异性涂色探针，通过种间染色体涂色，首次比较系统的构建了狐蝠科（3种）、菊头蝠科（6种）、蹄蝠科（4种）、假吸血蝠科（1种）、鞘尾蝠科（1种）和犬吻蝠科（2种）共6个科17种蝙蝠间的染色体同源图谱，并将它们与已发表的染色体图谱进行整合。我们的结果不仅揭示了这些物种间的核型进化关系及其核型进化的主要机制，而且加深了我们对整个翼手目系统发育关系的认识。研究结果表明： 1） 长舌果蝠亚科和狐蝠亚科的果蝠部分物种的核型（2n = 36）可能代表了整个狐蝠科的祖先形式，而狐蝠亚科的犬蝠部分物种的核型属于衍生形式。2） 整臂保守性广泛存在于菊头蝠属物种的核型进化中，罗伯逊易位是其核型进化的主要方式。此外，整臂的相互易位也可能在其中起一定的作用。 3） 蹄蝠科核型进化的主要机制为罗伯逊易位和臂内倒位。而臂内倒位和异染色质扩增在蹄蝠属的物种形成中可能起了非常重要的作用。 4） 与蹄蝠属相比，三叶蹄蝠属物种的核型中可能包含更多的祖先染色体或染色体片段；中蹄蝠与大蹄蝠具有较近的亲缘关系；与中蹄蝠和大蹄蝠相比，小蹄蝠的核型较为原始。 5） 犬吻蝠科内来自不同属但具有相同二倍染色体数目的物种间核型非常保守；该科物种核型间的差异主要是由倒位和异染色质的分布引起的。 6） 菊头蝠超科的菊头蝠科、蹄蝠科和假吸血蝠科具有较近的亲缘关系，并且它们与大蝙蝠亚目的狐蝠科共有一些细胞遗传学特征，进一步支持了小蝙蝠亚目双系起源的观点。

翼手目四个类群的比较分子细胞遗传学研究

翼手类(Order Chiroptera)是现生哺乳动物中唯一真正具有空中飞翔能力的动物；同时，它也是哺乳动物中为数不多的具有回声定位功能的几个分类类群之一。现存的翼手目动物约有1100种，占整个哺乳动物种类的20％以上，是哺乳动物中仅次于啮齿目的第二大目。尽管对于翼手目系统发育已经进行了大量的形态学和分子系统学等方面研究，但是翼手目中主要类群的系统发育关系尚存在着许多争议。进化过程中发生的染色体重排在揭示物种的系统发育关系中起到非常重要的作用。种间染色体涂色可以准确、快速、直观地鉴定物种进化过程中发生的大规模染色体重排，已成为在全基因组水平上比较研究哺乳动物核型多样性及其起源和演化的首选方法。利用染色体涂色可以构建不同物种间的比较染色体图谱，通过分析进化过程中保守的染色体片段在不同类群物种核型中的分布和排列方式，可以推导各类群可能的祖先核型，并重建伴随物种形成所发生的基因组变化历史，包括染色体重排的类型、速率和核型演化的趋势。但是，目前关于翼手目染色体涂色研究还很少，至今仍不能为翼手目各级分类水平上的系统发育关系研究提供系统完整的细胞遗传学证据。本研究利用大鼠耳蝠染色体特异涂色探针，通过种间染色体涂色，首次构建了狐蝠科、菊头蝠科、蹄蝠科和蝙蝠科代表物种间的比较染色体同源图谱，并且将它们与之前发表的用人的染色体探针构建的比较染色体图谱进行整合。通过分析这些物种之间保守染色体片段的分布，推测翼手目动物在核型进化过程中所发生的染色体重排的类型和数量，以期为研究翼手目的系统发育关系提供细胞遗传学证据。研究结果表明： 1. 翼手目动物的大多数染色体臂具有高度保守性，罗伯逊易位是其核型进化的主要机制。此外，倒位也是翼手目染色体进化过程中常见的染色体重排方式。 2. 小蝙蝠亚目的菊头蝠科和蹄蝠科与大蝙蝠亚目的狐蝠科亲缘关系更为接近，而与小蝙蝠亚目其它科的亲缘关系较远。 3. 菊头蝠科和蹄蝠科应该各自作为独立的科，但这两个科拥有一个共同的细胞遗传学衍生特征，这一特征在翼手目其它科中并未发现，表明它们之间有非常紧密的系统发育关系。 4. 菊头蝠科的祖先核型可能并不是前人所推测的全由端着丝粒染色体构成的2n＝62的核型；很有可能是包含有双臂染色体的，2n低于62的核型。 5. 三叶小蹄蝠比中蹄蝠保留更多的祖先染色体特征，其核型蹄蝠科中较为原始的核型。 6. 着丝粒融合是蝙蝠科物种核型演化的主要机制。蝙蝠科的祖先核型并不是类似于2n=44鼠耳蝠的核型，而可能更类似于棕蝠的2n＝50核型，由全端着丝粒染色体构成。 7. 除着丝粒融合外，异染色质的扩增也是蝙蝠科扁颅蝠属和山蝠属的染色体进化方式之一。 8. 绒山蝠与三种亚洲的伏翼共有两个同源染色体片段联合（MMY 8＋11和9＋13）,提示蝙蝠科的山蝠属和伏翼属有非常紧密的亲缘关系。 9. MMY 9 + 23和MMY 18＋19 这两个保守的同源染色体片段联合是蝙蝠科扁颅蝠属的细胞遗传学鉴定特征。 通过翼手目这些动物的染色体涂色研究，我们不但在全基因组的水平上揭示了翼手目几个主要类群代表动物的核型系统发育关系，而且所构建的比较染色体图谱将有助于基因组的序列信息从已测序物种（人）向序列信息较少的翼手目物种转移，这些研究结果也可以为翼手目的分类、物种鉴定、系统发育关系和生物医学研究（如SARS病毒）等提供基础性资料。

Phylogenetic studies of pantherine cats (Felidae) based on multiple genes, with novel application of nuclear beta-fibrinogen intron 7 to carnivores

The pantherine lineage of the cat family Felidae (order: Carnivora) includes five big cats of genus Panthera and a great many midsized cats known worldwide. Presumably because of their recent and rapid radiation, the evolutionary relationship among pantherines remains ambiguous. We provide an independent assessment of the evolutionary history of pantherine lineage using two complete mitochondrial (mt) genes (ND2 and ND4) and the nuclear beta-fibrmogen intron 7 gene, whose utility in carnivoran phylogeny was first explored. The available four mt (ND5, cytb, 12S, and 16SrRNA) and two nuclear (IRBP and TTR) sequence loci were also combined to reconstruct phylogeny of 14 closely related cat species. Our analyses of combined mt data (six genes; approximate to 3750 bp) and combined mt and nuclear data (nine genes; approximate to 6500 bp) obtained identical tree topologies, which were well-resolved and strongly supported for almost all nodes. Monophyly of Panthera genus in pantherine lineage was confirmed and interspecific affinities within this genus revealed a novel branching pattern, with P. tigris diverging first in Panthera genus, followed by P. onca, P. leo, and last two sister species P. pardus and P. uncia. In addition, close association of Neofelis nebulosa to Panthera, the phylogenetic redefinition of Otocolobus manil within the domestic cat group, and the relatedness of Acinonyx jubatus and Puma concolor were all important findings in the resulting phylogenies. The potential utilities of nine different genes for phylogenetic resolution of closely related pantherine species were also evaluated, with special interest in that of the novel nuclear beta-fibrinogen intron 7. (c) 2005 Elsevier Inc. All rights reserved.

Large Gene Family Expansions and Adaptive Evolution for Odorant and Gustatory Receptors in the Pea Aphid, Acyrthosiphon pisum

Gaining insight into the mechanisms of chemoreception in aphids is of primary importance for both integrative studies on the evolution of host plant specialization and applied research in pest control management because aphids rely on their sense of smell

Structural and functional characterization of the human CCR5 receptor in complex with HIV gp120 envelope glycoprotein and CD4 receptor by molecular modeling studies

The entry of human immunodeficiency virus (HIV) into cells depends on a sequential interaction of the gp120 envelope glycoprotein with the cellular receptors CD4 and members of the chemokine receptor family. The CC chemokine receptor CCR5 is such a receptor for several chemokines and a major coreceptor for the entry of R5 HIV type-1 (HIV-1) into cells. Although many studies focus on the interaction of CCR5 with HIV-1, the corresponding interaction sites in CCR5 and gp120 have not been matched. Here we used an approach combining protein structure modeling, docking and molecular dynamics simulation to build a series of structural models of the CCR5 in complexes with gp120 and CD4. Interactions such as hydrogen bonds, salt bridges and van der Waals contacts between CCR5 and gp120 were investigated. Three snapshots of CCR5-gp120-CD4 models revealed that the initial interactions of CCR5 with gp120 are involved in the negatively charged N-terminus (Nt) region of CCR5 and positively charged bridging sheet region of gp120. Further interactions occurred between extracellular loop2 (ECL2) of CCR5 and the base of V3 loop regions of gp120. These interactions may induce the conformational changes in gp120 and lead to the final entry of HIV into the cell. These results not only strongly support the two-step gp120-CCR5 binding mechanism, but also rationalize extensive biological data about the role of CCR5 in HIV-1 gp120 binding and entry, and may guide efforts to design novel inhibitors.

Bead-probe complex capture a couple of SINE and LINE family from genomes of two closely related species of East Asian cyprinid directly using magnetic separation

Background: Short and long interspersed elements (SINEs and LINEs, respectively), two types of retroposons, are active in shaping the architecture of genomes and powerful tools for studies of phylogeny and population biology. Here we developed special protocol to apply biotin-streptavidin bead system into isolation of interspersed repeated sequences rapidly and efficiently, in which SINEs and LINEs were captured directly from digested genomic DNA by hybridization to bead-probe complex in solution instead of traditional strategy including genomic library construction and screening. Results: A new couple of SINEs and LINEs that shared an almost identical 3'tail was isolated and characterized in silver carp and bighead carp of two closely related species. These SINEs (34 members), designated HAmo SINE family, were little divergent in sequence and flanked by obvious TSD indicated that HAmo SINE was very young family. The copy numbers of this family was estimated to 2 x 10(5) and 1.7 x 10(5) per haploid genome by Real-Time qPCR, respectively. The LINEs, identified as the homologs of LINE2 in other fishes, had a conserved primary sequence and secondary structures of the 3'tail region that was almost identical to that of HAmo SINE. These evidences suggest that HAmo SINEs are active and amplified recently utilizing the enzymatic machinery for retroposition of HAmoL2 through the recognition of higher-order structures of the conserved 42-tail region. We analyzed the possible structures of HAmo SINE that lead to successful amplification in genome and then deduced that HAmo SINE, SmaI SINE and FokI SINE that were similar in sequence each other, were probably generated independently and created by LINE family within the same lineage of a LINE phylogeny in the genomes of different hosts. Conclusion: The presented results show the advantage of the novel method for retroposons isolation and a pair of young SINE family and its partner LINE family in two carp fishes, which strengthened the hypotheses containing the slippage model for initiation of reverse transcription, retropositional parasitism of SINEs on LINEs, the formation of the stem loop structure in 3'tail region of some SINEs and LINEs and the mechanism of template switching in generating new SINE family.

Molecular evidence for diphyly of the Asian catfish family Amblycipitidae (Teleostei: Siluriformes) and exclusion of the South American Aspredinidae from Sisoroidea

Amblycipitidae Day, 1873 is an Asian family of catfishes (Siluriformes) usually considered to contain 28 species placed in three genera: Amblyceps (14 spp.), Liobagrus (12 spp.) and Xiurenbagrus (2 spp.). Morphology-based systematics has supported the monophyly of this family, with some authors placing Amblycipitidae within a larger group including Akysidae, Sisoridae and Aspredinidae, termed the Sisoroidea. Here we investigate the phylogenetic relationships among four species of Amblyceps, six species of Liobagrus and the two species of Xiurenbagrus with respect to other sisoroid taxa as well as other catfish groups using 6100 aligned base pairs of DNA sequence data from the rag1 and rag2 genes of the nuclear genome and from three regions (cyt b, COL ND4 plus tRNA-His and tRNA-Ser) of the mitochondrial genome. Parsimony and Bayesian analyses of the data indicate strong support for a diphyletic Amblycipitidae in which the genus Amblyceps is the sister group to the Sisoridae and a clade formed by genera Liobagrus and Xiurenbagrus is the sister group to Akysidae. These taxa together form a well supported monophyletic group that assembles all Asian sisoroid taxa, but excludes the South American Aspredinidae. Results for aspredinids are consistent with previous molecular studies that indicate these catfishes are not sisoroids, but the sister group to the South American doradoid catfishes (Auchenipteridae + Doradidae). The redefined sisoroid clade plus Bagridae, Horabagridae and (Ailia + Laides) make up a larger monophyletic group informally termed "Big Asia." Likelihood-based SH tests and Bayes Factor comparisons of the rag and the mitochondrial data partitions considered separately and combined reject both the hypothesis of amblycipitid monophyly and the hypothesis of aspredinid inclusion within Sisoroidea. This result for amblycipitids conflicts with a number of well documented morphological synapomorphies that we briefly review. Possible nomenclatural changes for amblycipitid taxa are noted.

Molecular phylogenetics of the family Cyprinidae (Actinopterygii : Cypriniformes) as evidenced by sequence variation in the first intron of S7 ribosomal protein-coding gene: Further evidence from a nuclear gene of the systematic chaos in the family

The family Cyprinidae is the largest freshwater fish group in the world, including over 200 genera and 2100 species. The phylogenetic relationships of major clades within this family are simply poorly understood, largely because of the overwhelming diversity of the group; however, several investigators have advanced different hypotheses of relationships that pre- and post-date the use of shared-derived characters as advocated through phylogenetic systematics. As expected, most previous investigations used morphological characters. Recently, mitochondrial DNA (mtDNA) sequences and combined morphological and mtDNA investigations have been used to explore and advance our understanding of species relationships and test monophyletic groupings. Limitations of these studies include limited taxon sampling and a strict reliance upon maternally inherited mtDNA variation. The present study is the first endeavor to recover the phylogenetic relationships of the 12 previously recognized monophyletic subfamilies within the Cyprinidae using newly sequenced nuclear DNA (nDNA) for over 50 species representing members of the different previously hypothesized subfamily and family groupings within the Cyprinidae and from other cypriniform families as outgroup taxa. Hypothesized phylogenetic relationships are constructed using maximum parsimony and Basyesian analyses of 1042 sites, of which 971 sites were variable and 790 were phylogenetically informative. Using other appropriate cypriniform taxa of the families Catostomidae (Myxocyprinus asiaticus), Gyrinocheilidae (Gyrinocheilus aymonieri), and Balitoridae (Nemacheilus sp. and Beaufortia kweichotvensis) as outgroups, the Cyprinidae is resolved as a monophyletic group. Within the family the genera Raiamas, Barilius, Danio, and Rasbora, representing many of the tropical cyprinids, represent basal members of the family. All other species can be classified into variably supported and resolved monophyletic lineages, depending upon analysis, that are consistent with or correspond to Barbini and Leuciscini. The Barbini includes taxa traditionally aligned with the subfamily Cyprininae sensu previous morphological revisionary studies by Howes (Barbinae, Labeoninae, Cyprininae and Schizothoracinae). The Leuciscini includes six other subfamilies that are mainly divided into three separate lineages. The relationships among genera and subfamilies are discussed as well as the possible origins of major lineages. (c) 2008 Published by Elsevier Inc.

Phylogenetic relationships of Danio within the order cypriniformes: A framework for comparative and evolutionary studies of a model species

The evolutionary relationships of species of Danio and the monophyly and phylogenetic placement of the genus within the family Cyprinidae and subfamily Rasborinae provide fundamentally important phyloinformatics necessary for direct evaluations of an array of pertinent questions in modern comparative biology. Although the genus Danio is not one of the most diverse within the family, Danio rerio is one of the most important model species in biology. Many investigations have used this species or presumed close relatives to address specific questions that have lasting impact on the hypothesis and theory of development in vertebrates. Largely lacking from this approach has been a holistic picture of the exact phylogenetic or evolutionary relationships of this species and its close relatives. One thing that has been learned over the previous century is that many organismal attributes (e.g., developmental pathways, ecologies, behaviors, speciation) are historically constrained and their origins and functions are best explained via a phylogenetic approach. Herein, we provide a molecular evaluation of the phylogenetic placement of the model species Danio rerio within the genus Danio and among hypothesized closely related species and genera. Our analysis is derived from data using two nuclear genes (RAG1, rhodopsin) and five mitochondrial genes (ND4, ND4L, ND5, COI, cyt b) evaluated using parsimony, maximum likelihood, and Bayesian analyses. The family Cyprinidae is resolved as monophyletic but the subfamily Rasborinae (priority over Danioinae) is an unnatural assemblage. Danio is identified as a monophyletic group sister to a clade inclusive of the genera Chela, Microrasbora, Devario, and Inlecypris, not Devario nor Esomus as hypothesized in previous studies. Danio rerio is sister to D. kyathit among the species of Danio evaluated in this analysis. Microrasbora and Rasbora are non-monophyletic assemblages; however, Boraras is monophyletic.

Identification of a novel C1q family member in color crucian carp (Carassius auratus) ovary

Potential roles of Clq/tumor necrosis factor (TNF) superfamily proteins have been observed in vertebrate oogenesis and oocyte maturation, but no ovary-specific member has been identified so far. In this study, we have cloned and identified a novel member of Clq family with a Clq domain in the C-terminal from fully grown oocyte cDNA library of color crucian carp and demonstrated that the gene might be specifically expressed in ovary and therefore designated as Carassius auratus ovary-specific Clq-like factor, CaOClq-like factor. It encodes a 213 amino acid protein with a 17 amino acid signal peptide. There is only one protein band of about 24.5 kDa in the extracts from phase I to phase IV oocytes, but two positive protein bands are detected in the extracts of mature eggs and fertilized eggs. Furthermore, the mobility shift of the smaller target protein band cannot be eliminated by phosphatase treatment, but the larger protein band increases its mobility on the gel after phosphatase treatment, suggesting that the larger protein might be a phosphorylated form. Immunofluorescence localization indicates that the CaOClq-like proteins localize in cytoplasm, cytoplasm membrane and egg envelope of the oocytes at cortical granule stage and vitellogenesis stage, whereas they were compressed to cytoplasm margin in ovulated mature eggs and discharged into perivitelline space between cytoplasm membrane and egg envelope after egg fertilization. Further studies on distribution and translocation mechanism of the CaOClq-like factor will be benefit to elucidate the unique function in oogenesis, oocyte maturation and egg fertilization. (C) 2004 Elsevier Inc. All rights reserved.