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.     

Melon bacterial artificial chromosome (BAC) library construction using improved methods and identification of clones linked to the locus conferring resistance to melon Fusarium wilt (Fom-2).

Utilizing improved methods, two bacterial artificial chromosome (BAC) libraries were constructed for the multidisease-resistant line of melon MR-1. The HindIII library consists of 177 microtiter plates in a 384-well format, while the EcoRI library consists of 222 microtiter plates. Approximately 95.6% of the HindIII library clones contain nuclear DNA inserts with an average size of 118 kb, providing a coverage of 15.4 genome equivalents. Similarly, 96% of the EcoRI library clones contain nuclear DNA inserts with an average size of 114 kb, providing a coverage of 18.7 genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBac536 vector, and organellar DNA sequences. High-density filters were screened with two genetic markers FM and AM that co-segregate with Fom-2, a gene conferring resistance to races 0 and 1 of Fusarium wilt. Fourteen and 18 candidate BAC clones were identified for the FM and AM probes, respectively, from the HindIII library, while 34 were identified for the AM probe from filters A, B, and C of the EcoRI library.     

Construction of tomato genomic DNA libraries in a binary-BAC (BIBAC) vector

This is the first report of large insert genomic DNA libraries constructed in a binary-BAC (BIBAC) vector. Genomic DNA libraries containing approximately 4.6 haploid nuclear genomic equivalents were constructed for Lycopersicon esculentum (cv. Mogeor) and Lycopersicon pennellii (LA716) in the BIBAC2 vector. The L. esculentum library has an average insert size of 125 kb and is comprised of 42 272 individual colonies stored as frozen cultures in a 384-well format (108 plates). The L. pennellii library has an average insert size of 90 kb and is comprised of 53 760 individual clones (140 384-well plates). In each of the libraries, it is estimated that 90% of the colonies contain genomic DNA inserts. The composition of the L. esculentum and L. pennellii libraries was determined by analyzing a series of randomly selected clones. The L. esculentum library was surveyed for clones containing chloroplast DNA (1.4%), mitochondrial DNA (0.012%) and repetitive DNA motifs. BIBAC clones that may contain a gene of interest can be identified from these libraries by colony hybridization with homologous or heterologous probes or by PCR pooling techniques. Once identified, BIBAC genomic DNA library clones are immediately suitable for Agrobacterium tumefaciens-mediated plant transformation.     

Picoeukaryotic Plankton Diversity at the Helgoland Time Series Site as Assessed by Three Molecular Methods

We analyzed picoeukaryote assemblages in the German Bight at the Helgoland time series site by sequencing cloned eukaryotic 18S rRNA genes in six genetic libraries plus one library from the Orkney Islands from a cruise of opportunity. The libraries were constructed from environmental samples collected at different periods of the year. The same samples were also analyzed using a fingerprinting technique, single-strand conformational polymorphism (SSCP), and DNA microarrays with class-level oligonucleotide probes. One hundred unique clones were analyzed from each library, thus insuring over 85% coverage of the library. The V4 region of the 18S rRNA gene was sequenced from each of these clones, thus providing the most discrimination among the clones. The nonphotosynthetic picoeukaryotic component dominated over the photosynthetic one and was represented by the ciliates at 45% and group II alveolates at 42%. Prasinophytes dominated the photosynthetic group at 40%, but other picoplankton groups, such as bolidomonads and chrysophytes, were also present. Totally novel groups were found in the cryptomonads and in the dinoflagellates. A new algal group sister to the cryptophyte nuclear gene and the glaucocystophytes was also found. These three groups have been found in other picoeukaryotic planktonic clone libraries. SSCP analyses at closer time intervals suggest that clone libraries should be made at weekly intervals if succession in the picoeukaryotic plankton community is to be monitored accurately. A comparison of annual samples suggests thatthere appears to be an annual cycle with regard to species composition. Microarray analysis supported the clone library data and offered a faster means of community analysis, which can be performed with similar accuracy and with higher throughput for a more in-depth analysis.     

Construction of BAC and BIBAC libraries from sunflower and identification of linkage group-specific clones by overgo hybridization

Complementary BAC and BIBAC libraries were constructed from nuclear DNA of sunflower cultivar HA 89. The BAC library, constructed with BamHI in the pECBAC1 vector, contains 107,136 clones and has an average insert size of 140 kb. The BIBAC library was constructed with HindIII in the plant-transformation-competent binary vector pCLD04541 and contains 84,864 clones, with an average insert size of 137 kb. The two libraries combined contain 192,000 clones and are equivalent to approximately 8.9 haploid genomes of sunflower (3,000 Mb/1C), and provide a greater than 99% probability of obtaining a clone of interest. The frequencies of BAC and BIBAC clones carrying chloroplast or mitochondrial DNA sequences were estimated to be 2.35 and 0.04%, respectively, and insert-empty clones were less than 0.5%. To facilitate chromosome engineering and anchor the sunflower genetic map to its chromosomes, one to three single- or low-copy RFLP markers from each linkage group of sunflower were used to design pairs of overlapping oligonucleotides (overgos). Thirty-six overgos were designed and pooled as probes to screen a subset (5.1×) of the BAC and BIBAC libraries. Of the 36 overgos, 33 (92%) gave at least one positive clone and 3 (8%) failed to hit any clone. As a result, 195 BAC and BIBAC clones representing 19 linkage groups were identified, including 76 BAC clones and 119 BIBAC clones, further verifying the genome coverage and utility of the libraries. These BAC and BIBAC libraries and linkage group-specific clones provide resources essential for comprehensive research of the sunflower genome.     

Construction of Highly Extensive Polymorphic DNA Libraries by in-Gel Competitive Reassociation Procedure

Differential genomic DNA libraries between two mouse strains and from two human individuals were constructed by means of the in-gel competitive reassociation (IGCR) procedure, a procedure developed for cloning altered anonymous restriction fragments. The libraries were highly enriched in RFLP fragments, approximately 60 and 40% for the mouse and human libraries, respectively, and, more importantly, maintained most of the original complexities of the RFLP fragments. Therefore, differential genomic DNA libraries constructed by the IGCR procedure, particularly for human genomic DNA, should offer highly extensive sources for polymorphic DNA sequences necessary for a variety of genome analyses, including studies on the origin and mechanism of biological diversity among the same species.     

Metagenomic analysis of Streptomyces lividans reveals host-dependent functional expression

Most functional metagenomic studies have been limited by the poor expression of many genes derived from metagenomic DNA in Escherichia coli, which has been the predominant surrogate host to date. To expand the range of expressed genes, we developed tools for construction and functional screening of metagenomic libraries in Streptomyces lividans. We expanded on previously published protocols by constructing a system that enables retrieval and characterization of the metagenomic DNA from biologically active clones. To test the functionality of these methods, we constructed and screened two metagenomic libraries in S. lividans. One was constructed with pooled DNA from 14 bacterial isolates cultured from Alaskan soil and the second with DNA directly extracted from the same soil. Functional screening of these libraries identified numerous clones with hemolytic activity, one clone that produces melanin by a previously unknown mechanism, and one that induces the overproduction of a secondary metabolite native to S. lividans. All bioactive clones were functional in S. lividans but not in E. coli, demonstrating the advantages of screening metagenomic libraries in more than one host.     

Construction of two BAC libraries from cucumber (Cucumis sativus L.) and identification of clones linked to yield component quantitative trait loci

Two bacterial artificial chromosome (BAC) libraries were constructed from an inbred line derived from a cultivar of cucumber (Cucumis sativus L.). Intact nuclei were isolated and embedded in agarose plugs, and high-molecular-weight DNA was subsequently partially digested with BamHI or EcoRI. Ligation of double size-selected DNA fragments with the pECBAC1 vector yielded two libraries containing 23,040 BamHI and 18,432 EcoRI clones. The average BamHI and EcoRI insert sizes were estimated to be 107.0 kb and 100.8 kb, respectively, and BAC clones lacking inserts were 1.3% and 14.5% in the BamHI and EcoRI libraries, respectively. The two libraries together represent approximately 10.8 haploid cucumber genomes. Hybridization with a C₀t-1 DNA probe revealed that approximately 36% of BAC clones likely carried repetitive sequence-enriched DNA. The frequencies of BAC clones that carry chloroplast or mitochondrial DNA range from 0.20% to 0.47%. Four sequence-characterized amplified region (SCAR), four simple sequence repeat, and an randomly amplified polymorphic DNA marker linked with yield component quantitative trait loci were used either as probes to hybridize high-density colony filters prepared from both libraries or as primers to screen an ordered array of pooled BAC DNA prepared from the BamHI library. Positive BAC clones were identified in predicted numbers, as screening by polymerase chain reaction amplification effectively overcame the problems associated with an overabundance of positives from hybridization with two SCAR markers. The BAC clones identified herein that are linked to the de (determinate habit) and F (gynoecy) locus will be useful for positional cloning of these economically important genes. These BAC libraries will also facilitate physical mapping of the cucumber genome and comparative genome analyses with other plant species.     

Construction and analysis of DNA sequence libraries from flow-sorted chromosomes: practical and theoretical considerations.

We describe the construction and analysis of recombinant DNA libraries representative of chromosomes 1 and 2 of Chinese hamster (Cricetulus griseus). Propidium-iodide stained chromosomes were purified by flow cytometric analysis and sorting, and EcoRI digests of purified DNA were cloned into the bacteriophage vector Charon 4A. These libraries contain DNA complementary to 63% and 69% of nick-translated DNA derived from flow-purified chromosomes 1 and 2, respectively. However, sequences complementary to only 24% and 35% of a total Chinese hamster genomic DNA tracer were hybridized in parallel renaturation experiments. The chromosome 2 library contained DNA sequences encoding dihydrofolate reductase (dhfr), a gene previously mapped to Chinese hamster chromosome 2. No sequences complementary to dhfr were found in the library constructed from chromosome 1 DNA. These analyses are discussed with regard to the current limitations and future strategies for the construction of chromosome-specific DNA sequence libraries of high purity and completeness.     

Development of a Nuclear Export Signal Trapping Method for Isolating Genes with HIV Rev Activity

We have developed a method for nuclear export signal trapping (NEST) to isolate functional Rev clones from various types of libraries such as libraries of Rev mutants. The expression libraries are cotransfected into COS cells together with a novel Rev-dependent immunoselectable CD28 expression plasmid, pCMV128-CD28. CD28-positive cells are recovered by FACS or by immune precipitation with magnetic beads, and the low-molecular-weight extra chromosomal DNA is recovered, amplified for Rev-containing DNA by PCR and recloned into expression plasmids. The resulting clones are enriched for functional Rev clones. These can be recovered efficiently after several repetitive NEST cycles. This technique may be usefully applied to study various regions of Rev, such as the RNA binding domain and the nuclear export signal, or effector domain and potentially to the isolation of cellular factors with nuclear export capabilities.     

Construction and Characterization of Two Bacterial Artificial Chromosome Libraries of Grass Carp

Bacterial artificial chromosome (BAC) library is an important tool in genomic research. We constructed two libraries from the genomic DNA of grass carp (Ctenopharyngodon idellus) as a crucial part of the grass carp genome project. The libraries were constructed in the EcoRI and HindIII sites of the vector CopyControl pCC1BAC. The EcoRI library comprised 53,000 positive clones, and approximately 99.94% of the clones contained grass carp nuclear DNA inserts (average size, 139.7 kb) covering 7.4× haploid genome equivalents and 2% empty clones. Similarly, the HindIII library comprised 52,216 clones with approximately 99.82% probability of finding any genomic fragments containing single-copy genes; the average insert size was 121.5 kb with 2.8% insert-empty clones, thus providing genome coverage of 6.3× haploid genome equivalents of grass carp. We selected gene-specific probes for screening the target gene clones in the HindIII library. In all, we obtained 31 positive clones, which were identified for every gene, with an average of 6.2 BAC clones per gene probe. Thus, we succeeded in constructing the desired BAC libraries, which should provide an important foundation for future physical mapping and whole-genome sequencing in grass carp.     

Isolation and characterization of the highly repeated fraction of the banana genome

Although the nuclear genome of banana (Musa spp.) is relatively small (1C approximately 610 Mbp for M. acuminata), the results obtained from other sequenced genomes suggest that more than half of the banana genome may be composed of repetitive and non-coding DNA sequences. Knowledge of repetitive DNA can facilitate mapping of important traits, phylogenetic studies, BAC-based physical mapping, and genome sequencing/annotation. However, only a few repetitive DNA sequences have been characterized in banana. In this work, we used DNA reassociation kinetics to isolate the highly repeated fraction of the banana genome (M. acuminata 'Calcutta 4'). Two libraries, one prepared from Cot 相似文献   

Construction and characterization of bacterial artificial chromosome libraries from the silkworm, Bombyx mori

Two bacterial artificial chromosome (BAC) libraries were constructed using nuclear DNA from posterior silkglands of the silkworm (Bombyx mori) strains p50 and C108. The libraries contain a total of 36,864 clones, or approximately 9 genome equivalents. The average insert sizes in the libraries were 134.5?kb and 120.8?kb, respectively. PCR-based screening was performed on the p50 library using probes for 34 sequence-tagged sites (STSs). Between 3 and 11 (6.1 hits on average) clones were isolated with each STS, in good agreement with the library size, 5.8 genome equivalents. The previously reported close linkage between the Bombyx homologs of the invected (Bm in) and engrailed (Bm en) genes was confirmed by construction of a BAC contig that contained both. Moreover, screening revealed novel information about the chromosomal organization of the sericin-1 and DH-PBAN genes, which were localized within a 22-kb interval and are divergently oriented. These results show that it is possible to construct contigs and analyze chromosome organization using these libraries.     

Fungal diversity in sheep (Ovis aries) and cattle (Bos taurus) feces assessed by comparison of 18S, 28S and ITS ribosomal regions

To explore the fungal diversity in ruminant feces for bioenergy, libraries based on internal transcribed spacer (ITS), 18S rRNA, and 28S rRNA regions were constructed, respectively. Although the libraries were constructed from the same DNA extracts, the fungal taxa analyses based on these libraries are different. The ITS and 28S libraries comprised higher proportions of fungal clones than 18S libraries, and the ITS libraries converged into the lower diversities. The ITS libraries could be used to analyze the fungal community. The 18S libraries were suitable for the fungi and protozoa community. However, the 28S are suitable for analysis of Ascomycota fungi. The major fungal taxa in cattle feces analyzed by ITS, 18S, and 28S libraries are similar to those of sheep feces, respectively. The fungal taxa detected by the ITS library comprised only 20 % fungal taxa detected by the three libraries. The 18S library comprised 30 % fungal taxa; the 28S library comprised about 50 % fungal taxa. The results indicated that primer sets toward different DNA regions lead to the difference in structures of fungal community. So the selection of primer sets may influence the fungal communities, and libraries based on single primer sets may underestimate the fungal diversity. The comparison of ITS, 18S, and 28S libraries could fid more diverse fungi than that based on only one library.     

宏基因组学在微生物活性物质筛选中的应用

采用传统分离培养筛选微生物新活性物质的方法受到很大制约,自然界99%以上的微生物不能培养,其资源开发受到很大限制。环境微生物宏基因组技术应用避开了微生物分离纯培养问题,极大拓展了微生物资源的利用空间,增加获得新活性物质的机会和途径。本文着重介绍宏基因组的概念、研究策略包括DNA提取、文库构建与筛选等及在微生物活性物质筛选中的应用,并对宏基因组研究中存在的问题进行探讨。     

一种用于构建红色红曲霉基因组Cosmid文库的大片段DNA制备方法

摘要：【目的】制备用于构建红色红曲霉cosmid文库的大片段基因组DNA。【方法】采用优化的酚氯仿抽提法制备DNA,并利用Sau3AI切割至平均大小为40 kb,然后使用Stratagene包装蛋白构建cosmid文库。基于PCR法使用同源探针从该文库中进行了目的基因的筛选。【结果】制备了浓度为5 μg/μL,平均片段大小大于48 kb的红色红曲霉大片段基因组DNA。利用该DNA构建的cosmid文库基因组覆盖倍数为10,并筛选到了含有目的片段的cosmid。【结论】通过该方法制备红色红曲霉大片段基因组D     

利用烟草基因组DNA构建近随机多肽文库

介绍一种新的方法构建近随机多肽文库。选取从大基因组物种的组织或细胞中提取的基因组DNA ,利用切割频率高的限制性内切酶切割 ,产生的短片段可以近似地认为是随机序列的片段 ,将它们与匹配的载体连接后转化进宿主细胞进行表达 ,从而获得近随机多肽文库。这样的文库可以用于蛋白质相互作用的研究。同一种基因组DNA可以利用不同的酶切 ,再分别连接到表达载体的不同读码框架 ,从而产生不同编码序列的多种近随机多肽文库。介绍了充分利用烟草基因组DNA构建两种不同酶切 ,三种读码框架 ,共六种不同编码序列的近随机多肽文库的方法。     

Tobacco nuclear DNA contains long tracts of homology to chloroplast DNA

Summary Long tracts of DNA with high sequence homology to chloroplast DNA were isolated from nuclear genomic libraries of Nicotiana tabacum. One lambda EMBL4 clone was characterised in detail and assigned to nuclear DNA. The majority of the 15.5-kb sequence is greater than 99% homologous with its chloroplast DNA counterpart, but a single base deletion causes premature termination of the reading frame of the psaA gene. One region of the clone contains a concentration of deleted regions, and these were used to identify and quantify the sequence in native nuclear DNA by polymerase chain reaction (PCR) methods. An estimated 15 copies of this specific region are present in a 1c tobacco nucleus.     

Characterization of DNA sequences associated with residual nuclei of Saccharomyces cerevisiae

We have used two different approaches to determine whether particular DNA sequences are specifically associated with high-salt-treated residual nuclei of Saccharomyces cerevisiae. First, libraries of yeast DNA in phage lambda were probed with nick-translated total nuclear or residual nuclear DNA from unsynchronized yeast cells. None of the plaques gave a significantly stronger or weaker signal with the residual nuclear probe than with the total nuclear probe. Second, DNA was purified from whole nuclei or residual nuclei which had been isolated from cells in G1, G1/S, early S, or nuclear division. This DNA was "dot-blotted" and then probed with specific yeast DNA sequences. Ribosomal DNA was 2- to 3-fold enriched in residual nuclei in late G1, G1/S, and early S, and 2 microns plasmid DNA sequences were 3- to 5-fold depleted during nuclear division and early G1. However, ARS1, TRP1, CEN6, and a telomere sequence were neither enriched nor depleted at any time during the cell cycle.     

Using partial genomic fosmid libraries for sequencing complete organellar genomes

Organellar genome sequences provide numerous phylogenetic markers and yield insight into organellar function and molecular evolution. These genomes are much smaller in size than their nuclear counterparts; thus, their complete sequencing is much less expensive than total nuclear genome sequencing, making broader phylogenetic sampling feasible. However; for some organisms, it is challenging to isolate plastid DNA for sequencing using standard methods. To overcome these difficulties, we constructed partial genomic libraries from total DNA preparations of two heterotrophic and two autotrophic angiosperm species using fosmid vectors. We then used macroarray screening to isolate clones containing large fragments of plastid DNA. A minimum tiling path of clones comprising the entire genome sequence of each plastid was selected, and these clones were shotgun-sequenced and assembled into complete genomes. Although this method worked well for both heterotrophic and autotrophic plants, nuclear genome size had a dramatic effect on the proportion of screened clones containing plastid DNA and, consequently, the overall number of clones that must be screened to ensure full plastid genome coverage. This technique makes it possible to determine complete plastid genome sequences for organisms that defy other available organellar genome sequencing methods, especially those for which limited amounts of tissue are available.