Construction and characterization of bacterial artificial chromosome library of black-handed spider monkey (Ateles geoffroyi)

The large-insert genomic DNA library is a critical resource for genome-wide genetic dissection of target species. We constructed a high-redundancy bacterial artificial chromosome (BAC) library of a New World monkey species, the black-handed spider monkey

Construction of a BAC library for Chinese amphioxus Branchiostoma belcheri and identification of clones containing Amphi-pax genes

Amphioxus is a crucial organism for the study of vertebrate evolution. Although a genomic BAC library of Branchiostoma floridae has been constructed, we report here another BAC library construction of its distant relative species Branchiostoma belcheri. The amphioxus BAC library established in present study consists of 45,312 clones arrayed in one hundred and eighteen 384-well plates. The average insert fragment size was 120 kb estimated by Pulsed Field Gel Electrophoresis (PFGE) analysis of 318 randomly selected clones. The representation of the library is about 12 equivalent to the genome, allowing a 99.9995% probability of recovering any specific sequence of interest. We further screened the library with 4 single copied Amphi-Pax genes and identified total of 26 positive clones with average of 6.5 clones for each gene. The result indicates this library is well suited for many applications and should also serve as a useful complemental resource for the scientific community.

Construction, characterization and chromosomal mapping of bacterial artificial chromosome (BAC) library of Yunnan snub-nosed monkey (Rhinopithecus bieti)

We constructed a high redundancy bacterial artificial chromosome library of a seriously endangered Old World Monkey, the Yunnan snub-nosed monkey (Rhinopithecus bieti) from China. This library contains a total of 136 320 BAC clones. The average insert size of BAC clones was estimated to be 148 kb. The percentage of small inserts (50-100 kb) is 2.74%, and only 2.67% non-recombinant clones were observed. Assuming a similar genome size with closely related primate species, the Yunnan snub-nosed monkey BAC library has at least six times the genome coverage. By end sequencing of randomly selected BAC clones, we generated 201 sequence tags for the library. A total of 139 end-sequenced BAC clones were mapped onto the chromosomes of Yunnan snub-nosed monkey by fluorescence in-situ hybridization, demonstrating a high degree of synteny conservation between humans and Yunnan snub-nosed monkeys. Blast search against human genome showed a good correlation between the number of hit clones and the size of the chromosomes, an indication of unbiased chromosomal distribution of the BAC library. This library and the mapped BAC clones will serve as a valuable resource in comparative genomics studies and large-scale genome sequencing of nonhuman primates. The DNA sequence data reported in this paper were deposited in GenBank and assigned the accession number CG891489-CG891703.

Phylogeny of the specialized schizothoracine fishes (Teleostei : Cypriniformes : Cyprinidae)

Phylogeny of the specialized schizothoracine fishes (Teleostei: Cypriniformes: Cyprinidae). Zoological Studies 40(2). 147-157. To elucidate phylogenetic relationships within the specialized schizothoracine fishes, we used 41 variable osteological and exte

Molecular phylogeny of the specialized schizothoracine fishes (Teleostei : Cyprinidae), with their implications for the uplift of the Qinghai-Tibetan Plateau

Molecular phylogeny of three genera containing nine species and subspecies of the specialized schizothoracine fishes are investigated based on the complete nucleotide sequence of mitochondrial cytochrome b gene. Meantime relationships between the main cladogenetic events of the specialized schizothoracine fishes and the stepwise uplift of the Qinghai-Tibetan Plateau are also conducted using the molecular clock, which is calibrated by geological isolated events between the upper reaches of the Yellow River and the Qinghai Lake. Results indicated that the specialized schizothoracine fishes are not a monophyly. Five species and subspecies of Ptychobarbus form a monophyly. But three species of Gymnodiptychus do not form a monophyly. Gd. integrigymnatus is a sister taxon of the highly specialized schizothoracine fishes while Gd. pachycheilus has a close relation with Gd. dybowskii, and both of them are as a sister group of Diptychus maculatus. The specialized schizothoracines fishes might have originated during the Miocene (about 10 MaBP), and then the divergence of three genera happened during late Miocene (about 8 MaBP). Their main specialization occurred during the late Pliocene and Pleistocene (3.54-0.42 MaBP). The main cladogenetic events of the specialized schizothoracine fishes are mostly correlated with the geological tectonic events and intensive climate shift happened at 8, 3.6, 2.5 and 1.7 MaBP of the late Cenozoic. Molecular clock data do not support the hypothesis that the Qinghai-Tibetan Plateau uplifted to near present or even higher elevations during the Oligocene or Miocene, and neither in agreement with the view that the plateau uplifting reached only to an altitude of 2000 in during the late Pliocene (about 2.6 MaBP).

Construction and characterization of a Lipotes vexillifer genomic DNA BAC library

We constructed a genomic DNA library for Lipotes vexillifer (L. vexillifer), the Baiji or Yangtze River dolphin, one of the most endangered mammals in the world. The library consists of 149,000 BAC clones, with an average insert size of 83 kb, representing approximately 3.4 haploid genome equivalents. PCR amplification of four known L. vexillifer genes yielded two to four positive clones each. To demonstrate the utility of this library, we isolated and sequenced the L. vexillifer alpha lactalbumin gene, which is a gene specific to mammals and one which has been widely used as molecular tool in phylogenetic analysis. We also end-sequenced 20 randomly selected clones, resulting in the identification of at least five new L. vexilliter genes, five SSR loci, and one SINE locus. These results suggest that this library is a valuable resource for candidate gene cloning, physical mapping, and genome sequencing of this important and threatened species.

Molecular phylogeny and biogeography of the highly specialized grade schizothoracine fishes (Teleostei : Cyprinidae) inferred from cytochrome b sequences

We recovered the phylogenetic relationships among 23 species and subspecies of the highly specialized grade schizothoracine fishes distributing at 36 geographical sites in the Tibetan Plateau and its Surrounding regions by analyzing sequences of cytochrome b genes. Furthermore, we estimated the possible divergent times among lineages based on a historical geological isolation event in the Tibetan Plateau. The molecular data revealed that the highly specialized grade schizothoracine fishes were not a monophyletic group, but were the same as genera Gymnocypris and Schizogypsis. Our results indicated that the molecular phylogenetic relationships apparently reflected their geographical and historical associations with drainages, namely species from the same and adjacent drainages clustered together and had close relationships. The divergence times of different lineages were well consistent with the rapid uplift phases of the Tibetan Plateau in the late Cenozoic, suggesting that the origin and evolution of schizothoracine fishes were strongly influenced by environment changes resulting from the upheaval of the Tibetan Plateau.

A combined phage display ScFv library against Myxobolus rotundus infecting crucian carp, Carassius auratus auratus (L.), in China

Immunological methods have been developed for the diagnosis of Myxobolus rotundus but their use has been limited for the prevention and therapy of this serious parasitic pathogen. Phage display antibody libraries are a powerful technique for the development of antibodies to molecules of interest and have advantages over traditional hybridroma approaches. In the present study, four antigen fractions related to M. rotundus were prepared and a combined phage display single-chain antibody fragments (ScFv) library was constructed against this parasite. Preliminary analysis indicated that a combined antibody library of about 2.08 X 10(5) individual clones and high diversity was generated. After four rounds of screening (bio-panning) against soluble spore protein prepared from lysed, intact, mature M rotundus spores, a strain monoclonal phage display ScFv, termed pCAN-6H9, with better affinity, was isolated. The pCAN-6H9 gene fragment was sequenced and analysed. The specificity of pCAN-6H9 was further demonstrated by dot-blot. In competition enzyme-linked immunosorbent assay, both the original and enriched phage-displayed ScFv repertoire showed significant inhibition of mouse anti-M rotundus serum binding to coated antigen, while the inhibition rate of monoclonal pCAN-6H9 phage particles was only 11.83%.

Molecular phylogeny of the specialized schizothoracine fishes (Teleostei : Cyprinidae), with their implications for the uplift of the Qinghai-Tibetan Plateau

Molecular phylogeny of three genera containing nine species and subspecies of the specialized schizothoracine fishes are investigated based on the complete nucleotide sequence of mitochondrial cytochrome b gene. Meantime relationships between the main cladogenetic events of the specialized schizothoracine fishes and the stepwise uplift of the Qinghai-Tibetan Plateau are also conducted using the molecular clock, which is calibrated by geological isolated events between the upper reaches of the Yellow River and the Qinghai Lake. Results indicated that the specialized schizothoracine fishes are not a monophyly. Five species and subspecies of Ptychobarbus form a monophyly. But three species of Gymnodiptychus do not form a monophyly. Gd. integrigymnatus is a sister taxon of the highly specialized schizothoracine fishes while Gd. pachycheilus has a close relation with Gd. dybowskii, and both of them are as a sister group of Diptychus maculatus. The specialized schizothoracines fishes might have originated during the Miocene (about 10 MaBP), and then the divergence of three genera happened during late Miocene (about 8 MaBP). Their main specialization occurred during the late Pliocene and Pleistocene (3.54-0.42 MaBP). The main cladogenetic events of the specialized schizothoracine fishes are mostly correlated with the geological tectonic events and intensive climate shift happened at 8, 3.6, 2.5 and 1.7 MaBP of the late Cenozoic. Molecular clock data do not support the hypothesis that the Qinghai-Tibetan Plateau uplifted to near present or even higher elevations during the Oligocene or Miocene, and neither in agreement with the view that the plateau uplifting reached only to an altitude of 2000 in during the late Pliocene (about 2.6 MaBP).

Phylogeny of the specialized schizothoracine fishes (Teleostei : Cypriniformes : Cyprinidae)

Phylogeny of the specialized schizothoracine fishes (Teleostei: Cypriniformes: Cyprinidae). Zoological Studies 40(2). 147-157. To elucidate phylogenetic relationships within the specialized schizothoracine fishes, we used 41 variable osteological and external characters among this groups, three species of Schizothorax, and 1 fossil species. When the 3 species of Schizothorax were designated as an outgroup and all 41 characters were set as unordered with equal weighting, the data matrix yielded a single most-parsimonious tree with a tree length of 71 steps, a consistency index of 0.6761, and a retention index of 0.7416. Meanwhile, a bootstrap test was conducted to verify the reliability of the results. The matrix was also analyzed for different conditions: all characters were ordered and the fossil species was added as an outgroup. The phylogenetic analyses presented herein support the following hypotheses. 1) All species of the specialized schizo-thoracines fishes form a monophyletic group. 2) Monophyly of the genus Ptychobarbus is not supported by the bootstrap test or when these characters are ordered. 3) The genus Gymnodiptychus forms a monophyletic group. 4) All species of Ptychobarbus and Gymnodiptychus form a monophyletic group with Diptychus as its sister group.

Parallel synthesis and herbicidal activity of pyrethroid library

A pyridine-containing pyrethroid library of 255 compounds was conveniently constructed using parallel synthesis. The library was screened by high through-put screening (HTs), and further study was focused on the compounds with initial bioactivities. The results suggest that some compounds could be potential herbicides.

Efficient One-Pot Synthesis of Highly Substituted Thiophene Library from 1,3-Dicarbonyl Compounds

A faccile and efficient one-pot synthesis of highly substituted thiophenes has been developed and employed for the preparation of a small focused library. Treatment of 1,3-dicarbonyl compounds 1 with CS2 in the presence of K2CO3 in DMF at room temperature, followed by stepwise addition of alkyl bromides 2 and methylene active bromides 3, provided via intramolecular cyclization 2,3,4,5-tetrasubstituted thiophenes 4 in yields of 77-94%. This protocol, combining construction and modification of the thiophene ring, increases the structural diversity of final products from readily available materials. A mechanism for the one-pot synthesis of thiophenes of type 4 has been proposed. A small focused library of thiophenes is prepared using the sequential addition of reagents to achieve unique substitution in the 2 and 5 position of the thiophene ring