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 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.     

Development of an efficient maintenance and screening system for large-insert genomic DNA libraries of hexaploid wheat in a transformation-competent artificial chromosome (TAC) vector

Three large-insert genomic DNA libraries of common wheat, Triticum aestivum cv. Chinese Spring, were constructed in a newly developed transformation-competent artificial chromosome (TAC) vector, pYLTAC17, which accepts and maintains large genomic DNA fragments stably in both Escherichia coli and Agrobacterium tumefaciens. The vector contains the cis sequence required for Agrobacterium-mediated gene transfer into grasses. The average insert sizes of the three genomic libraries were approximately 46, 65 and 120 kbp, covering three haploid genome equivalents. Genomic libraries were stored as frozen cultures in a 96-well format, each well containing approximately 300-600 colonies (12 plates for small library, four for medium-size library and four for large library). In each of the libraries, approximately 80% of the colonies harbored genomic DNA inserts of >50 kbp. TAC clones containing gene(s) of interest were identified by the pooled PCR technique. Once the target TAC clones were isolated, they could be immediately transferred into grass genomes with the Agrobacterium system. Five clones containing the thionin type I genes (single copy per genome), corresponding to each of the three genomes (A, B and D), were successfully selected by the pooled PCR method, in addition to an STS marker (aWG464; single copy per genome) and CAB (a multigene family). TAC libraries constructed as described here can be used to isolate genomic clones containing target genes, and to carry out genome walking for positional cloning.     

Construction of a quinoa (Chenopodium quinoa Willd.) BAC library and its use in identifying genes encoding seed storage proteins

Quinoa (Chenopodium quinoa Willd.) is adapted to the harsh environments of the Andean Altiplano region. Its seeds have a well-balanced amino acid composition and exceptionally high protein content with respect to human nutrition. Quinoa grain is a staple in the diet of some of the most impoverished people in the world. The plant is an allotetraploid displaying disomic inheritance (2n=4x=36) with a di-haploid genome of 967 Mbp (megabase pair), or 2C=2.01 pg. We constructed two quinoa BAC libraries using BamHI (26,880 clones) and EcoRI (48,000 clones) restriction endonucleases. Cloned inserts in the BamHI library average 113 kb (kilobase) with approximately 2% of the clones lacking inserts, whereas cloned inserts in the EcoRI library average 130 kb and approximately 1% lack inserts. Three plastid genes used as probes of high-density arrayed blots of 73,728 BACs identified approximately 2.8% of the clones as containing plastid DNA inserts. We estimate that the combined quinoa libraries represent at least 9.0 di-haploid nuclear genome equivalents. An average of 12.2 positive clones per probe were identified with 13 quinoa single-copy ESTs as probes of the high-density arrayed blots, suggesting that the estimate of 9.0× coverage of the genome is conservative. Utility of the BAC libraries for gene identification was demonstrated by probing the library with a partial sequence of the 11S globulin seed storage protein gene and identifying multiple positive clones. The presence of the 11S globulin gene in four of the clones was verified by direct comparison with quinoa genomic DNA on a Southern blot. Besides serving as a useful tool for gene identification, the quinoa BAC libraries will be an important resource for physical mapping of the quinoa genome.     

Construction and characterization of two BAC libraries from Brachypodium distachyon, a new model for grass genomics.

Brachypodium is well suited as a model system for temperate grasses because of its compact genome and a range of biological features. In an effort to develop resources for genome research in this emerging model species, we constructed 2 bacterial artificial chromosome (BAC) libraries from an inbred diploid Brachypodium distachyon line, Bd21, using restriction enzymes HindIII and BamHI. A total of 73,728 clones (36,864 per BAC library) were picked and arrayed in 192,384-well plates. The average insert size for the BamHI and HindIII libraries is estimated to be 100 and 105 kb, respectively, and inserts of chloroplast origin account for 4.4% and 2.4%, respectively. The libraries individually represent 9.4- and 9.9-fold haploid genome equivalents with combined 19.3-fold genome coverage, based on a genome size of 355 Mb reported for the diploid Brachypodium, implying a 99.99% probability that any given specific sequence will be present in each library. Hybridization of the libraries with 8 starch biosynthesis genes was used to empirically evaluate this theoretical genome coverage; the frequency at which these genes were present in the library clones gave an estimated coverage of 11.6- and 19.6-fold genome equivalents. To obtain a first view of the sequence composition of the Brachypodium genome, 2185 BAC end sequences (BES) representing 1.3 Mb of random genomic sequence were compared with the NCBI GenBank database and the GIRI repeat database. Using a cutoff expectation value of E<10-10, only 3.3% of the BESs showed similarity to repetitive sequences in the existing database, whereas 40.0% had matches to the sequences in the EST database, suggesting that a considerable portion of the Brachypodium genome is likely transcribed. When the BESs were compared with individual EST databases, more matches hit wheat than maize, although their EST collections are of a similar size, further supporting the close relationship between Brachypodium and the Triticeae. Moreover, 122 BESs have significant matches to wheat ESTs mapped to individual chromosome bin positions. These BACs represent colinear regions containing the mapped wheat ESTs and would be useful in identifying additional markers for specific wheat chromosome regions.     

Generation of shrnas from randomized oligonucleotides

Suppression of gene expression by small interfering RNA (siRNA) has proved to be a gene-specific and cost effective alternative to other gene suppression technologies. Short hairpin RNAs (shRNAs) generated from the vector-based expression are believed to be processed into functional siRNAs in vivo, leading to gene silencing. Since an shRNA library carries a large pool of potential siRNAs, such a library makes it possible to knock down gene expression at the genome wide scale. Although much of research has been focused on generating shRNA libraries from either individually made gene specific sequences or cDNA libraries, there is no report on constructing randomized shRNA libraries, which could provide a good alternative to these existing libraries. We have developed a method of constructing shRNAs from randomized oligonucleotides. Through this method, one can generate a partially or fully randomized shRNA library for various functional analyses. We validated this procedure by constructing a p53-specific shRNA. Western blot revealed that the p53-shRNA successfully suppressed expression of the endogenous p53 in MCF-7 cells. We then made a partially randomized shRNA library. Sequencing of 15 randomly picked cloned confirmed the randomness of the library. Therefore, the library can be used for various functional assays, such as target validation when a suitable screening or selection method is available.     

Bacterial artificial chromosome (BAC) library resource for positional cloning of pest and disease resistance genes in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

Pest and disease problems are important constraints of cassava production and host plant resistance is the most efficient method of combating them. Breeding for host plant resistance is considerably slowed down by the crop’s biological constraints of a long growth cycle, high levels of heterozygosity and a large genetic load. More efficient methods such as gene cloning and transgenesis are required to deploy resistance genes. To facilitate the cloning of resistance genes, bacterial artificial chromosome (BAC) library resources have been developed for cassava. Two libraries were constructed from the cassava clones, TMS 30001, resistant to the cassava mosaic disease (CMD) and the cassava bacterial blight (CBB), and MECU72, resistant to cassava white fly. The TMS30001 library has 55 296 clones with an insert size range of 40–150 kb with an average of 80 kb, while the MECU72 library consists of 92 160 clones and an insert size range of 25–250 kb average of 93 kb. Based on a genome size of 772 Mb, the TMS30001 and MECU72 libraries have a 5 and 11.3 haploid genome equivalents and a 95 and 99 chance of finding any sequence, respectively. To demonstrate the potential of the libraries, the TMS30001 library was screened by southern hybridization using a cassava analog (CBB1) of the Xa21 gene from rice that maps to a region containing a QTL for resistance to CBB as probe. Five BAC clones that hybridized to CBB1 were isolated and a Hind III fingerprint revealed 2–3 copies of the gene in individual BAC clones. A larger scale analysis of resistance gene analogs (RGAs) in cassava has also been conducted in order to understand the number and organization of RGAs. To scan for gene and repeat DNA content in the libraries, end-sequencing was performed on 2301 clones from the MECU72 library. A total of 1705 unique sequences were obtained with an average size of 715 bp. Database homology searches using BLAST revealed that 458 sequences had significant homology with known proteins and 321 with transposable elements. The use of the library in positional cloning of pest and disease resistance genes is discussed.     

Construction and characterisation of a gridded chicken cosmid library with four-fold genomic coverage

Gridded genomic libraries are crucial for the positional candidate gene approach. For this purpose we constructed a gridded genomic library from a female chicken using the vector sCos 1. About 110 000 cosmid clones were grown and replicated in 384-well plates. An average insert size of 39 kb was calculated from the analysis of 68 randomly selected clones. No chimerism could be observed from 31 in situ hybridisations. One replica of the library (number 125) has been transferred to the Resource Centre/Primary Database (RZPD) of the German Human Genome Project (DHGP). The whole library was gridded onto four nylon filters at high density for efficient identification of cosmid clones by colony hybridisation. Twenty-two probes were used for screening the library and each of them gave at least one positive signal. This result is in good agreement with a four-fold coverage of the genome as estimated from the insert length and number of recombinant clones. This library provides a powerful tool for rapid physical mapping and complex analysis of the chicken genome.     

Transforming a set of biological flat file librariesto a fast access network

SRS (Sequence Retrieval System), an indexing system for flatfile libraries, provides fast access to individual library entriesvia retrie by keywords from rious data fields. SRS is now alsoable to build indices using cross–references that mostlibraries provide. Fifteen libraries of DNA and protein sequencesand structures have been selected. These libraries interactwith at least one other by means of cross–references.Indexing these cross–references allows a complete networkof libraries to be built. In the network an entry from one librarycan be linked in principle to every other library. If two librariesare not directly cross–referenced, the linkage can bemade with a succession of single links between neighbouring,cross–referenced libraries. A new operator has been addedto the query language of SRS for convenient specification oflinks amongst complete libraries or entry sets generated byprevious queries on particular libraries. All the informationin the network can now be used to retrieve an entry in a specificlibrary, e.g. the full information given in amino acid sequenceentries from SwissProt can now be used to retrieve related tertiarystructure entries from PDB. Furthermore, a search in a singlelibrary can be extended to a search in the complete librarynetwork, e.g. all entries in all databases pertaining to elastasecan be found.     

Identification of a gene encoding an immuno-reactive membrane protein from Campylobacter jejuni

A gene encoding a putative membrane protein has been identified from Campylobacter jejuni NCTC 11168 following an immuno-screen of a lambda ZAP II genomic DNA library with antiserum raised against glycine-extractable proteins. The nucleotide sequence of the entire genomic insert revealed six open reading frames, all but one of which have sequence homologues in the complete genome sequence of Helicobacter pylori. The gene encoding the immuno-reactive protein was further identified by independent expression of these reading frames in Escherichia coli. The gene encodes an integral membrane protein, expression of which in E. coli results in a profound filamentous phenotype.     

Construction of a Normalized cDNA Library for the Trypanosoma cruzi Genome Project

Sequencing of the Trypanosoma cruzi genome is underway. Expressed sequence tags, obtained from cDNA libraries, facilitate mapping and gene discovery. The efficiency of large-scale generation of such tags is increased when using normalized cDNA libraries, where the frequency of individual clones is brought within a narrow range. Repetitive sequencing of abundant clones is therefore minimized. We constructed a normalized cDNA library from epimastigotes of clone CL Brener, and the efficiency of normalization of representative clones was assessed and shown to be adequate. The normalized cDNA library has been distributed to several groups and large-scale sequencing is currently in progress.     

几种全长cDNA文库构建方法比较

全长cDNA文库是高效、大规模获得基因全序列信息的一条有效途径,尤其是对基因组庞大,近期内尚不能进行全基因组测序的生物来说,是一条开展功能基因组研究的重要途径。本文对几种全长cDNA文库的构建方法进行了概述,针对各种方法的原理及优缺点做了分析、比较,并结合本实验室的结果,重点介绍了Cap-trapper法在小麦全长cDNA文库中的应用及文库中全长基因比例判定方法。     

Construction and characterization of a bacterial artificial chromosome library ofArabidopsis thaliana

We constructed an ordered 3,948-clone arabidopsis BAC library. The library has a combined average insert size of 100 kb (n=54). Assuming a haploid genome size of 100,000 kb, the BAC library contains 3.95 haploid genome equivalents with a 98 percent probability of isolating a specific genomic region. The library was screened with five arabidopsis cDNA probes and one tomato probe; all probes hybridized to at least one (and in most cases three) BAC clones in the library.     

Sequence-Verified Two-Allele Transposon Mutant Library for Pseudomonas aeruginosa PAO1

Mutant hunts using comprehensive sequence-defined libraries make it possible to identify virtually all of the nonessential functions required for different bacterial processes. However, the success of such screening depends on the accuracy of mutant identification in the mutant library used. To provide a high-quality library for Pseudomonas aeruginosa PAO1, we created a sequence-verified collection of 9,437 transposon mutants that provides genome coverage and includes two mutants for most genes. Mutants were cherry-picked from a larger library, colony-purified, and resequenced both individually using Sanger sequencing and in a pool using Tn-seq. About 8% of the insertion assignments were corrected, and in the final library nearly 93% of the transposon locations were confirmed by at least one of the resequencing procedures. The extensive sequence verification and inclusion of more than one mutant for most genes should help minimize missed or erroneous genotype-phenotype assignments in studies using the new library.     

Analysis of three different repeated DNA elements present in the P1 operon of Mycoplasma pneumoniae: size, number and distribution on the genome.

Mycoplasma pneumoniae, a bacterium pathogenic for humans, has a relatively small genome size of 840 kbp. Even though, several repeated DNA elements have been identified in the genome of this prokaryote, particularly within the P1 gene which codes for a major adhesin protein of M. pneumoniae. These elements were characterized in detail with respect to size, number and distribution on the genome, represented by an ordered cloned library covering the complete chromosome. Three different repetitive elements were detected in and around the P1 gene designated as RepMP2/3, RepMP4 and RepMP5. The length of these elements varies between 1.1-1.5 kbp (RepMP4), 1.8 kbp (RepMP2/3) and 1.9-2.2 kpb (RepMP5). They occur at least 8 to 10 times on the chromosome. Possible functions are discussed and a uniform nomenclature for these repeats is proposed.     

Short reads, circular genome: skimming solid sequence to construct the bighorn sheep mitochondrial genome

As sequencing technology improves, an increasing number of projects aim to generate full genome sequence, even for nonmodel taxa. These projects may be feasibly conducted at lower read depths if the alignment can be aided by previously developed genomic resources from a closely related species. We investigated the feasibility of constructing a complete mitochondrial (mt) genome without preamplification or other targeting of the sequence. Here we present a full mt genome sequence (16,463 nucleotides) for the bighorn sheep (Ovis canadensis) generated though alignment of SOLiD short-read sequences to a reference genome. Average read depth was 1240, and each base was covered by at least 36 reads. We then conducted a phylogenomic analysis with 27 other bovid mitogenomes, which placed bighorn sheep firmly in the Ovis clade. These results show that it is possible to generate a complete mitogenome by skimming a low-coverage genomic sequencing library. This technique will become increasingly applicable as the number of taxa with some level of genome sequence rises.     

Development of new transformation-competent artificial chromosome vectors and rice genomic libraries for efficient gene cloning

The transformation-competent artificial chromosome vector (TAC) system has been shown to be very useful for efficient gene isolation in Arabidopsis thaliana (Proc. Natl. Acad. Sci. USA 96 (1998) 6535). To adapt the vector system for gene isolation in crops, two new TAC vectors and rice genomic libraries were developed. The new vectors pYLTAC17 and pYLTAC27 use the Bar gene and Hpt gene driven by the rice Act1 promoter as the plant selectable markers, respectively, and are suitable for transformation of rice and other grasses. Two representative genomic libraries (I and II) of an Indica rice variety Minghui63, a fertility restorer line for hybrid rice, were constructed with pYLTAC17 using different size classes of partially digested DNA fragments. Library I and library II consisted of 34,560 and 1.2 x 10(5) clones, with average insert sizes of approximately 77 and 39 kb, respectively. The genome coverage of the libraries I and II was estimated to be about 5 and 11 haploid genome equivalents, respectively. Clones of the library I were stored individually in ninety 384-well plates, and those of the library II were collected as bulked pools each containing 30-50 clones and stored in eight 384-well plates. A number of probes were used to hybridize high-density colony filters of the library I prepared by an improved replicating method and each detected 2-9 positive clones. A method for rapid screening of the library II by pooled colony hybridization was developed. A TAC clone having an 80 kb rice DNA insert was successfully transferred into rice genome via Agrobacterium-mediated transformation. The new vectors and the genomic libraries should be useful for gene cloning and genetic engineering in rice and other crops.     

乳酸乳球菌AL2基因文库的构建及乳链菌肽生物合成基因簇的筛选

用本实验室获得的乳链菌肽高产菌株乳酸乳球菌AL2总DNA为供体,以λ EMBL3为载体,构建了该菌株的基因文库,共获得3900多个噬斑。通过Southern杂交、PCR扩增及DNA序列测定,证实从该文库中筛选到一个含有完整乳链菌肽生物合成基因簇的重组噬菌体λHJ\|3。