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, 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.

Molecular characterization and expression profiles in response to bacterial infection of Chinese soft-shelled turtle interleukin-8 (IL-8), the first reptilian chemokine gene

In this study, an IL-8 homologue has been cloned and identified from a reptile, Chinese soft-shelled turtle for the first time. The full-length cDNA of turtle IL-8 was 1188 bp and contained a 312 bp open reading frame (ORF) coding for a protein of 104 amino acids. The chemokine CXC domain, which contained Glu-Leu-Arg (ELR) motif and four cysteine residues, was well conserved in turtle IL-8. The 4924 bp genomic DNA of turtle IL-8 contained four exons and three introns. Phylogenetic analysis showed that the amino acid sequence of turtle IL-8 clustered together with birds. RT-PCR analysis showed that turtle IL-8 mRNA was constitutively expressed liver, spleen, kidney, heart, blood and intestine tissues of control turtles. Real-time quantitative PCR analysis further indicated that the turtle IL-8 mRNA expression was apparent in various tissues at 8 h and up-regulated significantly during 8 h-7 d after Aeromonas hydrophila infection. The present studies will help us to understand the evolution of IL-8 molecule and the inflammatory response mechanism in reptiles. (C) 2009 Elsevier Ltd. All rights reserved.

Cloning of biologically active genomes from a Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus isolate by using a bacterial artificial chromosome

Purification of genotypes from baculovirus isolates provides understanding of the diversity of baculoviruses and may lead to the development of better pesticides. Here, we report the cloning of different genotypes from an isolate of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) by using a bacterial artificial chromosome (BAC). A transfer vector (pHZB10) was constructed which contained an Escherichia coli mini-F replicon cassette within the upstream and downstream arms of HaSNPV polyhedrin gene. Hz2e5 cells were co-transfected with wild-type HaSNPV DNA and pHZB10 to generate recombinant viruses by homologous recombination. The DNA of budded viruses (BVs) was used to transform E. coli. One of the bacmid colonies, HaBacHZ8, has restriction enzyme digestion profiles similar to an in vivo cloned strain HaSNPV-G4, the genome of which has been completely sequenced. For testing the oral infectivity, the polyhedrin gene of HaSNPV was reintroduced into HaBacHZ8 to generate the recombinant bacmid HaBacDF6. The results of one-step growth curves, electron microscopic examination, protein expression analysis and bioassays indicated that HaBacDF6 replicated as well as HaSNPV-G4 in vitro and in vivo. The biologically functional HaSNPV bacmids obtained in this research will facilitate future studies on the function genomics and genetic modification of HaSNPV. (C) 2003 Elsevier B.V. All rights reserved.

Atomic force microscope investigation of large-circle DNA molecules

A circular bacterial artificial chromosome of 148.9 kbp on human chromosome 3 has been extended and fixed on bare mica substrates using a developed fluid capillary flow method in evaporating liquid drops. Extended circular DNA molecules were imaged with an atomic force microscope (AFM) under ambient conditions. The measured total lengths of the whole DNA molecules were in agreement with sequencing analysis data with an error range of +/-3.6%. This work is important groundwork for probing single nucleotide polymorphisms in the human genome, mapping genomic DNA, manipulating biomolecular nanotechnology, and studying the interaction of DNA-protein complexes investigated by AFM.

Particle-attached and free-living bacterial communities in a contrasting marine environment: Victoria Harbor, Hong Kong

Diversity of particle-attached and free-living marine bacteria in Victoria Harbor, Hong Kong, and its adjacent coastal and estuarial environments was investigated using DNA fingerprinting and clone library analysis. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes showed that bacterial communities in three stations of Victoria Harbor were similar, but differed from those in adjacent coastal and estuarine stations. Particle-attached and free-living bacterial community composition differed in the Victoria Harbor area. DNA sequencing of 28 bands from DGGE gel showed Alphaproteobacteria was the most abundant group, followed by the Bacteroidetes, and other Proteobacteria. Bacterial species richness (number of DGGE bands) differed among stations and populations (particle-attached and free-living; bottom and surface). BIOENV analysis indicated that the concentrations of suspended solids were the major contributing parameter for the spatial variation of total bacterial community structure. Samples from representative stations were selected for clone library (548 clones) construction and their phylogenetic distributions were similar to those of sequences from DGGE. Approximately 80% of clones were affiliated to Proteobacteria, Bacteroidetes and Cyanobacteria. The possible influences of dynamic pollution and hydrological conditions in the Victoria Harbor area on the particle-attached and free-living bacterial community structures were discussed.

Particle-attached and free-living bacterial communities in a contrasting marine environment: Victoria Harbor, Hong Kong

Diversity of particle-attached and free-living marine bacteria in Victoria Harbor, Hong Kong, and its adjacent coastal and estuarial environments was investigated using DNA fingerprinting and clone library analysis. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes showed that bacterial communities in three stations of Victoria Harbor were similar, but differed from those in adjacent coastal and estuarine stations. Particle-attached and free-living bacterial community composition differed in the Victoria Harbor area. DNA sequencing of 28 bands from DGGE gel showed Alphaproteobacteria was the most abundant group, followed by the Bacteroidetes, and other Proteobacteria. Bacterial species richness (number of DGGE bands) differed among stations and populations (particle-attached and free-living; bottom and surface). BIOENV analysis indicated that the concentrations of suspended solids were the major contributing parameter for the spatial variation of total bacterial community structure. Samples from representative stations were selected for clone library (548 clones) construction and their phylogenetic distributions were similar to those of sequences from DGGE. Approximately 80% of clones were affiliated to Proteobacteria, Bacteroidetes and Cyanobacteria. The possible influences of dynamic pollution and hydrological conditions in the Victoria Harbor area on the particle-attached and free-living bacterial community structures were discussed.

Construction and characterization of a bacterial artificial chromosome library of marine macroalga Porphyra yezoensis (Rhodophyta)

A large-DNA-fragment library is necessary for research into the Porphyra genome. In this study, a bacterial artificial chromosome (BAC) library of Porphyra yezoensis was constructed and characterized. The library contains 54,144 BAC clones with an average insert size of about 65 kb and fewer than 0.7% of clones without large inserts. Therefore, its capacity is more than 6.6 P. yezoensis genome equivalents, and the probability of recovering any nuclear DNA sequence from the library is higher than 99%. The library shows good fidelity and stability. A putative trehalose-6-phosphate synthase (TPS) gene was successfully screened out from the library. The above results show that the library is useful for gene cloning and genomic research in P. yezoensis.

Vertical distribution of bacterial and archaeal communities along discrete layers of a deep-sea cold sediment sample at the East Pacific Rise (similar to 13 degrees N)

The community structure and vertical distribution of prokaryotes in a deep-sea (ca. 3,191 m) cold sediment sample (ca. 43 cm long) collected at the East Pacific Rise (EPR) similar to 13 degrees N were studied with 16SrDNA-based molecular analyses. Total community DNA was extracted from each of four discrete layers EPRDS-1, -2, -3 and -4 (from top to bottom) and 16S rDNA were amplified by PCR. Cluster analysis of DGGE profiles revealed that the bacterial communities shifted sharply between EPRDS-1 and EPRDS-2 in similarity coefficient at merely 49%. Twenty-three sequences retrieved from DGGE bands fell into 11 groups based on BLAST and bootstrap analysis. The dominant groups in the bacterial communities were Chloroflexi, Gamma proteobacteria, Actinobacterium and unidentified bacteria, with their corresponding percentages varying along discrete layers. Pairwise Fst (F-statistics) values between the archaeal clone libraries indicated that the archaeal communities changed distinctly between EPRDS-2 and EPRDS-3. Sequences from the archaeal libraries were divided to eight groups. Crenarchaea Marine Group I (MGI) was prevalent in EPRDS-1 at 83%, while Uncultured Crenarchaea group II B (UCII B) abounded in EPRDS-4 at 61%. Our results revealed that the vertically stratified distribution of prokaryotic communities might be in response to the geochemical settings and suggested that the sampling area was influenced by hydrothermalism. The copresence of members related to hydrothermalism and cold deep-sea environments in the microbial community indicated that the area might be a transitional region from hydrothermal vents to cold deep-sea sediments.

Construction and characterization of two bacterial artificial chromosome libraries of Zhikong scallop, Chlamys farreri Jones et Preston, and identification of BAC clones containing the genes involved in its innate immune system

Large-insert bacterial artificial chromosome (BAC) libraries are necessary for advanced genetics and genomics research. To facilitate gene cloning and characterization, genome analysis, and physical mapping of scallop, two BAC libraries were constructed from nuclear DNA of Zhikong scallop, Chlamys farreri Jones et Preston. The libraries were constructed in the BamHI and MboI sites of the vector pECBAC1, respectively. The BamHI library consists of 73,728 clones, and approximately 99% of the clones contain scallop nuclear DNA inserts with an average size of 110 kb, covering 8.0x haploid genome equivalents. Similarly, the MboI library consists of 7680 clones, with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1x. The combined libraries collectively contain a total of 81,408 BAC clones arrayed in 212 384-well microtiter plates, representing 9.1x haploid genome equivalents and having a probability of greater than 99% of discovering at least one positive clone with a single-copy sequence. High-density clone filters prepared from a subset of the two libraries were screened with nine pairs of Overgos designed from the cDNA or DNA sequences of six genes involved in the innate immune system of mollusks. Positive clones were identified for every gene, with an average of 5.3 BAC clones per gene probe. These results suggest that the two scallop BAC libraries provide useful tools for gene cloning, genome physical mapping, and large-scale sequencing in the species.

Construction of bacterial artificial chromosome libraries for Zhikong Scallop Chlamys farreri

Two Large-insert genomic bacterial artificial chromosome (BAC) libraries of Zhikong scallop Chlamys farreri were constructed to promote our genetic and genomic research. High-quality megabase-sized DNA was isolated from the adductor muscle of the scallop and partially digested by BamH I and Mbo I, respectively. The BamH I library consisted of 53 760 clones while the Mbo I library consisted of 7 680clones. Approximately 96 % of the clones in BamH I library contained nuclear DNA inserts in average size of 100 kb, providing a coverage of 5.3 haploid genome equivalents. Similarly, the Mbo I library with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1 haploid genome equivalents.

Identification and analysis of the immune effects of CpG motifs that protect Japanese flounder (Paralichthys olivaceus) against bacterial infection

CpG-containing oligodeoxynucleotides (ODNs) are known to be immunostimulatory in vertebrate systems and can activate both innate and adaptive immune responses. In this report, we described the selection, identification, and analysis of CpG motifs with immunoprotective effects in Japanese flounder. Sixteen CpG ODNs were synthesized and examined for the ability to inhibit bacterial dissemination in Japanese flounder blood. Four ODNs with the strongest inhibitory effects were selected and mixed to form ODNs 4M. In addition, a plasmid, pCN6, was constructed that contains the sequences of the four selected ODNs. When administered into Japanese flounder via intraperitoneal injection, both ODNs 4M and pCN6 could, in dose and time dependent manners, afford short-term protection against the infections of two different bacterial pathogens. Immunological analyses showed that ODNs 4M and, especially, pCN6 activated head kidney macrophages and enhanced serum bactericidal activity via probably the alternative pathway of complement activation. When used as a DNA vaccine to immunize Japanese flounder, pCN6 conferred apparent protections (42.9% and 52.6%, respectively, in terms of relative percent survival) against the challenges of two different fish pathogens at 4-week post-vaccination. Transcriptional analysis showed that vaccination with pCN6 upregulated the expression of the genes encoding NKEF, MHC II alpha, IL-1 beta, Mx, and MHC I alpha. These results demonstrate that ODNs 4M and pCN6 are immunostimulatory in Japanese flounder and can induce short- and long-term nonspecific protections against bacterial infections. (C) 2010 Elsevier Ltd. All rights reserved.