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.

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 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.

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.

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.

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.

中国穿山甲细菌人工染色体文库的构建及其特征鉴定

鳞甲目 （Pholidota）作为真兽亚纲18个现生目之一，是理解哺乳动物系统分类和进化的一个关键类群。穿山甲的形态十分特殊，它的系统发生地位历来就是人们关注的对象，搞清穿山甲的系统发生地位将有助于理解和重建整个哺乳动物的系统发生过程。目前，中国穿山甲已被列入“哺乳动物基因组计划”24个待测序物种其中之一。细胞遗传学的研究表明中国穿山甲的核型具有多态性。 细菌人工染色体 （BAC）文库目前已成为基因组学研究领域的最为重要的研究工具之一。各种动植物细菌人工染色体文库的逐渐丰富为后基因组时代比较基因组研究奠定了良好的基础。中国穿山甲基因组DNA文库的构建将有力地促进穿山甲染色体演化、哺乳动物比较基因组学及其系统发育学的研究。 本研究在世界上首次成功地构建了一个具有约8倍基因组覆盖率的中国穿山甲细菌人工染色体文库。该基因组文库含有208,512个克隆，储存在543块384孔板中。对随机选取的171个克隆的Not1酶切和脉冲场电泳结果分析显示: 64.5%的克隆插入片段大小介于100-149 Kb之间， 75.6%的克隆插入片段大于100kb。平均插入片段大小为111.4Kb; 非重组克隆 （无插入片段）的比率为9.9%。同时，我们通过荧光原位杂交把105个BAC克隆定位到了中国穿山甲染色体上的特异区域, 构建了中国穿山甲的BAC细胞遗传学图谱。初步的染色体定位结果表明，BAC克隆在基因组中的分布没有偏向性。 中国穿山甲BAC文库的成功构建，丰富了哺乳动物基因组文库资源。该文库除有利于比较基因组研究之外，也为深化保护遗传学研究奠定了基础。BAC细胞遗传学图谱的构建，将为中国穿山甲全基因组测序中产生的序列重叠群在染色体上的定向和定位奠定基础。

Genomic organization, gene duplication, and expression analysis of interleukin-1 beta in channel catfish (Ictalurus punctatus)

Interleukin-1 beta (IL-1 beta) is one of the pivotal early response pro-inflammatory cytokines that enables organisms to respond to infection and induces a cascade of reactions leading to inflammation. In spite of its importance and two decades of studies in the mammalian species, genes encoding IL-1 beta were not identified from non-mammalian species until recently. Recent research, particularly with genomic approaches, has led to sequencing of IL-1 beta from many species. Clinical studies also Suggested IL-1 beta as an immunoreagulatory molecule potentially useful for enhancing vaccination. However, no IL-1 beta genes have been identified from channel catfish, the primary aquaculture species from the United States. In this study, we identified two distinct cDNAs encoding catfish IL-1 beta. Their encoding genes were identified, sequenced, and characterized. The catfish IL-1 beta genes were assigned to bacterial artificial chromosome (BAC) clones. Genomic studies indicated that the IL-1 beta genes were tandemly duplicated on the same chromosome. Phylogenetic analysis of various IL-1 beta genes indicated the possibility of recent species-specific gene duplications in channel catfish, and perhaps also in swine and carp. Expression analysis indicated that both IL-1 beta genes were expressed, but exhibited distinct expression profiles in various catfish tissues, and after bacterial infection with Edwardsiella ictaluri. (c) 2005 Elsevier Ltd. All rights reserved.

滇金丝猴细菌人工染色体文库构建及Neurotrypsin基因的分子进化研究

本文包括两方面有关灵长类的研究工作。一是构建了一个非人灵长类（滇金丝猴）的人工染色体文库（BAC文库），为进一步研究灵长类的基因组进化奠定了一定的基础，二是对一个认知相关基因neuotrypsin在灵长类中的分子进化进行了详尽的研究，以期从分子进化角度认识该基因在灵长类高级认知功能发展中所起的作用。滇金丝猴（Rhinopithecusbieti），一个处于严重濒危状态的旧大陆猴，中国特产灵长类之一。在系统发育上它占据着旧大陆猴与小猿的中间地位，是研究灵长类进化的一个重要物种。，在本研究中，我们构建了一个滇金丝猴的细菌人工染色体文库（BAC文库）。该文库含有136320个BAC克隆，平均插入片段大小为148kb，小片段（50-100kb）所占的比例为2.74％，非重组克隆仅占2.67％。假定金丝猴与亲缘关系较近的灵长类有着相似的基因组大小，该文库至少有6倍的基因组覆盖率。对随机选取的BAC克隆进行末端测序，我们获得了201个序列标签（STS）。通过荧光原位杂交（FISH）技术，139个经末端测序的BAC克隆被精确定位到滇金丝猴的染色体上。荧光杂交实验还表明了人和金丝猴染色体产存在着高度的同线保守性。在人类基因组数据库中的Blast搜寻的结果显示染色体上的克隆数目与染色体大小呈很好的相关性，表明该文库克隆比较均匀地覆盖了滇金丝猴的基因组。该金丝猴文库及所定位的克隆将会成为非人灵长类的比较基因组研究和大规模基因组测序的一个宝贵的资源。Neurotrypsin是与个主要在脑中表达的，同神经发育和认知功能相关的胞外丝氨酸蛋白酶。它在人体中发生的突变与常染色体上隐性的非综合性精神发育迟缓（MR）紧密相关。我们通过对n个非人灵长类，包括大猿、小猿、旧大陆猴和新大陆猴neurotrypsin的编码区的测序，研究了neurotrypsin在灵长类中的分子进化。结果显示出，在灵长类进化过程中，neurotrypsin保持着由纯化性选择（负沟选择）所致的强烈的功能限制，这暗示着neurotrypsin在灵长类的认知发展中起着关键的功能性作用。进一步的分析表明，纯化性选择实际上只作用于neurotrypsin的介导其结合到其它细胞表面或胞外蛋白质的SRCR功能结构域上。另外，通过灵长类和其它哺乳动物目的比较，我们还发现，在鼠neuotrypsin中，一个人SRCR结构域（外显子2和3）的缺失是由于在鼠科动物中所发生的片段丢失事件所致。

扇贝、对虾基因组BAC文库构建及其重要功能基因的初步筛选和分析

基因组大片段BAC文库是进行生物遗传学和基因组学研究必不可少的基础工具。为了深入开展栉孔扇贝（Chlamys farreri）和凡纳滨对虾（Litopenaeus vannamei）基因组学研究、阐明其基因组的结构与功能、图位克隆重要功能基因、构建高密度物理图谱并最终实现与已有遗传连锁图的整合，本研究在植物基因组BAC文库构建技术的基础上，针对海洋生物的特点进行了大胆的改革与尝试，最终成功构建了C. ferrari和L. vannamei两种重要海水养殖动物的基因组BAC文库。 本文构建的栉孔扇贝基因组BAC文库，由BamHI文库和MboI文库构成。其中BamHI文库含有73,728个BAC克隆，空载率约为1%，平均插入片段约为110 kb，覆盖栉孔扇贝单倍体基因组约8倍；MboI文库共有7,680个克隆组成，平均插入片段大小约为145 kb，插入率为100%，覆盖栉孔扇贝单倍体基因组约1.1倍。两个栉孔扇贝基因组BAC文库共由81,408个克隆组成，平均插入片段约为113 kb，覆盖率约为栉孔扇贝单倍体基因组大小的9.1倍。 将栉孔扇贝基因组BAC文库的192个384微孔培养板中的73,728个BAC克隆以4 x 4点阵形式制备了高密度DNA薄膜，用于对感兴趣的基因及DNA序列的筛选。高密度DNA薄膜的覆盖率约为栉孔扇贝单倍体基因组的8.3倍。针对栉孔扇贝先天免疫系统通路的6个重要功能基因，根据栉孔扇贝cDNA序列以及异缘物种DNA序列设计了Overgo探针。利用Overgo探针对高密度DNA薄膜杂交筛选的结果显示，平均每个基因检测到7.3个潜在阳性克隆。 本研究所构建的凡纳滨对虾基因组HindIII酶切BAC文库共有102,528个BAC克隆，存放于267个384微孔培养板中，平均插入片段大小约为101 kb，空载率约为5%，覆盖L. vannamei单倍体基因组约5倍。将其中240个384微孔培养板中的92,160个BAC克隆以4 x 4的矩阵排列形式制作了5张高密度凡纳滨对虾DNA薄膜，约覆盖整个对虾单倍体基因组的4.5倍。针对6个与对虾免疫、生殖生理有关的重要功能基因设计了Overgo探针，杂交筛选出20个阳性克隆，平均每个基因有3.3个潜在阳性克隆。 以上筛选结果不仅为进一步研究这些功能基因的结构与功能、表达与调控，揭示它们在扇贝和对虾以及其他近缘种的免疫系统、抗逆和生殖生理过程中的作用机理打下了基础，同时也间接验证了栉孔扇贝和凡纳滨对虾基因组BAC文库将成为基因筛选、基因的结构与功能分析、基因图位克隆、物理图谱构建以及大规模全基因组测序等方面的有力工具。

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 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.