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

Diffusive coevolution and mutualism maintenance mechanisms in a fig-fig wasp system

In reciprocal mutualism systems, the exploitation events by exploiters might disrupt the reciprocal mutualism, wherein one exploiter species might even exclude other coexisting exploiter species over an evolutionary time frame. What remains unclear is how such a community is maintained. Niche partitioning, or spatial heterogeneity among the mutualists and exploiters, is generally believed to enable stability within a mutualistic system. However, our examination of a reciprocal mutualism between a fig species (Ficus racemosa) and its pollinator wasp (Ceratosolen fusciceps) shows that spatial niche partitioning does not sufficiently prevent exploiters from overexploiting the common resource (i.e., the female flowers), because of the considerable niche overlap between the mutualists and exploiters. In response to an exploiter, our experiment shows that the fig can (1) abort syconia-containing flowers that have been galled by the exploiter, Apocryptophagus testacea, which oviposits before the pollinators do; and (2) retain syconia-containing flowers galled by Apocryptophagus mayri, which oviposit later than pollinators. However, as a result of (2), there is decreased development of adult non-pollinators or pollinator species in syconia that have not been sufficiently pollinated, but not aborted. Such discriminative abortion of figs or reduction in offspring development of exploiters while rewarding cooperative individuals with higher offspring development by the fig will increase the fitness of cooperative pollinating wasps, but decrease the fitness of exploiters. The fig fig wasp interactions are diffusively coevolved, a case in which fig wasps diversify their genotype, phenotype, or behavior as a result of competition between wasps, while figs diverge their strategies to facilitate the evolution of cooperative fig waps or lessen the detrimental behavior by associated fig wasps. In habitats or syconia that suffer overexploitation, discriminative abortion of figs or reduction in the offspring development of exploiters in syconia that are not or not sufficiently pollinated will decrease exploiter fitness and perhaps even drive the population of exploiters to local extinction, enabling the evolution and maintenance of cooperative pollinators through the movement between habitats or syconia (i.e., the metapopulations).

Co-expression of CD173 (H2) and CD174 (Lewis Y) with CD44 suggests that fucosylated histo-blood group antigens are markers of breast cancer-initiating cells

Histo-blood group antigens CD173 (H2) and CD174 (Lewis Y) are known to be developmentally regulated carbohydrate antigens which are expressed to a varying degree on many human carcinomas. We hypothesized that they might represent markers of cancer-initiating cells (or cancer stem cells, CSC). In order to test this hypothesis, we examined the co-expression of CD173 and CD174 with stem cell markers CD44 and CD133 by flow cytometry analysis, immunocytochemistry, and immunohistochemistry on cell lines and tissue sections from breast cancer. In three breast cancer cell lines, the percentage of CD173(+)/CD44(+) cells ranged from 17% to > 60% and of CD174(+)/CD44(+) from 21% to 57%. In breast cancer tissue sections from 15 patients, up to 50% of tumor cells simultaneously expressed CD173, CD174, and CD44 antigens. Co-expression of CD173 and CD174 with CD133 was also observed, but to a lesser percentage. Co-immunoprecipitation and sandwich ELISA experiments on breast cancer cell lines suggested that CD173 and CD174 are carried on the CD44 molecule. The results show that in these tissues CD173 (H2) and CD174 (LeY) are associated with CD44 expression, suggesting that these carbohydrate antigens are markers of cancer-initiating cells or of early progenitors of breast carcinomas.

Comparative genomic analysis links karyotypic evolution with genomic evolution in the Indian Muntjac (Muntiacus muntjak vaginalis)

The karyotype of Indian muntjacs (Muntiacus muntjak vaginalis) has been greatly shaped by chromosomal fusion, which leads to its lowest diploid number among the extant known mammals. We present, here, comparative results based on draft sequences of 37 bac

Sequencing, annotation and comparative analysis of nine BACs of giant panda (Ailuropoda melanoleuca)

A 10-fold BAC library for giant panda was constructed and nine BACs were selected to generate finish sequences. These BACs could be used as a validation resource for the de novo assembly accuracy of the whole genome shotgun sequencing reads of giant panda newly generated by the Illumina GA sequencing technology. Complete sanger sequencing, assembly, annotation and comparative analysis were carried out on the selected BACs of a joint length 878 kb. Homologue search and de novo prediction methods were used to annotate genes and repeats. Twelve protein coding genes were predicted, seven of which could be functionally annotated. The seven genes have an average gene size of about 41 kb, an average coding size of about 1.2 kb and an average exon number of 6 per gene. Besides, seven tRNA genes were found. About 27 percent of the BAC sequence is composed of repeats. A phylogenetic tree was constructed using neighbor-join algorithm across five species, including giant panda, human, dog, cat and mouse, which reconfirms dog as the most related species to giant panda. Our results provide detailed sequence and structure information for new genes and repeats of giant panda, which will be helpful for further studies on the giant panda.

Evolutionary Conservation of Dazl Genomic Organization and its Continuous and Dynamic Distribution Throughout Germline Development in Gynogenetic Gibel Carp

To investigate germline development and germ cell specification, we identified a Dazl homolog (CagDazl) from gynogenetic gibel carp (Carassius auratus gibelio). Its cDNA sequence and BAC clone sequence analyses revealed the genomic organization conservation and conserved synteny of the Dazl family members and their neighborhood genes among vertebrates, especially in fish. Moreover, a polyclonal antibody specific to CagDazl was produced and used to examine its expression and distribution throughout germline development at protein level. Firstly, ovary-specific expression pattern of CagDazl was confirmed in adult tissues by RT-PCR and Western blot. In addition, in situ hybridization and immunofluorescence localization demonstrated its specific expression in germ cells, and both its transcript and protein were localized to germ plasm. Then, co-localization of CagDazl and mitochondrial cloud was found, confirming that CagDazl transcript and its protein are germ plasm component and move via METRO pathway during oogenesis. Furthermore, the CagDazl is abundant and continuous throughout germline development and germ cell specification including primordial germ cell (PGC) formation, oogonium differentiation, oocyte development, and embryogenesis, and the dynamic distribution occurs at different development stages. The data suggest that maternal CagDazl might play an important role in gibel carp PGC formation. Therefore, CagDazl is a useful and specific marker for tracing germ plasm and germ cell development in the gynogenetic gibel carp. In addition, in comparison with previous studies in sexual reproduction species, the continuous and dynamic distribution of CagDazl protein in the germ plasm throughout the life cycle seems to have significant implication in sex evolution of vertebrates. J. Exp. Zool. (Mol. Deu. Euol.) 312B:855-871, 2009. (C) 2009 Wiley-Liss, Inc.

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.

NK-lysin of channel catfish: Gene triplication, sequence variation, and expression analysis

Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. In addition to the previously known four classes of antimicrobial peptides, a fifth class of antimicrobial peptides has been recently identified to include NK-lysins that have a globular three-dimensional structure and are larger with 74-78 amino acid residues. NK-lysin has been shown to harbor antimicrobial activities against a wide spectrum of microorganisms including bacteria, fungi, protozoa, and parasites. To date, NK-lysin genes have been reported from only a limited number of organisms. We previously identified a NK-lysin cDNA in channel catfish. Here we report the identification of two noveltypes of NK-lysin transcripts in channel catfish. Altogether, three distinct NK-lysin transcripts exist in channel catfish. In this work, their encoding genes were identified, sequenced, and characterized. We provide strong evidence that the catfish NK-lysin gene is tripled in the same genomic neighborhood. All three catfish NK-lysin genes are present in the same genomic region and are tightly linked on the same chromosome, as the same BAC clones harbor all three copies of the NK-lysin genes. All three NK-lysin genes are expressed, but exhibit distinct expression profiles in various tissues. In spite of the existence of a single copy of NK-lysin gene in the human genome, and only a single hit from the pufferfish,genome, there are two tripled clusters of NK-lysin genes on chromosome 17 of zebrafish in addition to one more copy on its chromosome 5. The similarity in the genomic arrangement of the tripled NK-lysin genes in channel catfish and zebrafish suggest similar evolution of NK-lysin genes. (c) 2005 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.

DNA的扫描探针显微镜和电化学研究

1、在云母表面用AFM观察了不同离子对DNA形态影响。一些离子，如Mg~(2+)、Mn~(2+)、Co~(2+)等是固定和铺展DNA的较好介质，但缺陷是它们可以催化限制性内切酶对DNA的切割。在另一些离子存在下，如Co(phen)_3~(3+)和Ca~(2+)，限制酶不对DNA切割。Co(phen)_3~(3+)存在下，限制性内切酶EcoRI的星号活力位点与DNA结合。AFM直接观察到EcoRI在pBR322DNA上的星号活力图。AFM测得的星号活力物理图与理论偏差小于100 bp。获得了适于AFM观察的常规DNA铺展方法。长度大于40微米的DNA在几微米扫描范围内均匀铺展，并且云母基底表面平整干净。此铺展方法对DNA长度没有影响。这种展开的长链DNA适合于人类基因组BAC DNA的高分辨物理图制作。2、用低电流STM首次观察到金表面上自组装的ssDNA。ssDNA在局部规则区域内彼此平行排列。ssDNA链宽约为0.9纳米，其值为dsDNA的一半。在同一ssDNA链上，相邻两个亮点的距离为0.3-0.5纳米，其值与理论的ssDNA内相邻两碱基之间距离一致。电化学实验证明了自组装在Au（111）上的分子是ssDNA而不是dsDNA。讨论了传统基底-HOPG对生物样品的固定。溶菌酶被稳定地固定在氨基修饰的HOPG表面。通过低电流STM，我们观察到溶菌酶的外貌。3、侧向力显微镜对多头绒泡菌染色体的观察，我们获得了中期、前期染色体的表面精细结构和染色体的集缩过程形态。在染色体表面存在300和40纳米的细丝结构。讨论了零度扫描角的侧向力显微镜。4、研究了dsDNA和ssDNA在聚吡咯修饰电极表面的吸附行为，并通过电化学指示剂Co(phen)_3~(3+)在聚吡咯电极上区分了ssDNA和dsDNA。聚吡咯膜内掺杂的正电荷可以很好地吸附DNA分子，并且这种膜可以耐受一些变性条件的处理。再生这种电极是可能的。这些结果表明，这种新型DNA固定方法可以用于DNA传感器的研制。

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。

Band crossing in isovalent semiconductor alloys with large size mismatch: First-principles calculations of the electronic structure of Bi and N incorporated GaAs

For large size- and chemical-mismatched isovalent semiconductor alloys, such as N and Bi substitution on As sites in GaAs, isovalent defect levels or defect bands are introduced. The evolution of the defect states as a function of the alloy concentration is usually described by the popular phenomenological band anticrossing (BAC) model. Using first-principles band-structure calculations we show that at the impurity limit the N-(Bi)-induced impurity level is above (below) the conduction- (valence-) band edge of GaAs. These trends reverse at high concentration, i.e., the conduction-band edge of GaAs1-xNx becomes an N-derived state and the valence-band edge of GaAs1-xBix becomes a Bi-derived state, as expected from their band characters. We show that this band crossing phenomenon cannot be described by the popular BAC model but can be naturally explained by a simple band broadening picture.

甲醇的生物降解与含甲醇废水生物处理工艺研究

甲醇作为一种有机污染物广泛存在于有机合成和石油化工等生产废水中.该研究针对氮肥生产过程中产生的低浓度甲醇废水缺乏有效回收利用工艺的现实情况，以氮肥生产过程中产生的工艺冷凝液和尿素水解水为对象，通过甲醇高效降解菌的分离、固定化，生物活性炭（BAC）反应系统的建立，小试、中试等研究工作，确定了将其回用到脱盐水系统的可行性，提出了工艺参数和工业化方案，并对BAC生物膜结构特点进行了较为深入的研究.

宁康霉素对植物病原真菌的抑制作用

宁康霉素是由海洋细菌BAC-9912发酵获得的生物农药,对多种植物病原真菌有良好的抑制作用。本研究发现宁康霉素强烈抑制玉米纹枯病菌和苹果轮纹病菌的菌丝,可使玉米纹枯病菌菌丝枯萎和产生畸形。比较宁康霉素与农利灵和速克灵对玉米纹枯病菌的抑菌效果,结果显示宁康霉素的抑菌效果优于两种化学农药。

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。