Systematic sequencing of cDNA clones using the transposon Tn5

In parallel with the production of genomic sequence data, attention is being focused on the generation of comprehensive cDNA-sequence resources. Such efforts are increasingly emphasizing the production of high-accuracy sequence corresponding to the entire insert of cDNA clones, especially those presumed to reflect the full-length mRNA. The complete sequencing of cDNA clones on a large scale presents unique challenges because of the generally small, yet heterogeneous, sizes of the cloned inserts. We have developed a strategy for high-throughput sequencing of cDNA clones using the transposon Tn5. This approach has been tailored for implementation within an existing large-scale ‘shotgun-style’ sequencing program, although it could be readily adapted for use in virtually any sequencing environment. In addition, we have developed a modified version of our strategy that can be applied to cDNA clones with large cloning vectors, thereby overcoming a potential limitation of transposon-based approaches. Here we describe the details of our cDNA-sequencing pipeline, including a summary of the experience in sequencing more than 4200 cDNA clones to produce more than 8 million base pairs of high-accuracy cDNA sequence. These data provide both convincing evidence that the insertion of Tn5 into cDNA clones is sufficiently random for its effective use in large-scale cDNA sequencing as well as interesting insight about the sequence context preferred for insertion by Tn5.     

Expression sequence tag-specific full-length cDNA cloning: actin cDNAs

Current strategies for cDNA cloning are based on construction of cDNA libraries and colony screening. The process of obtaining a full-length cDNA clone can be highly time and labor intensive. Using the human actin gene as a model target cDNA, we have developed an RNA-capture method for rapid cloning of full-length cDNAs. The approach involves the capture of mRNA with expressed sequence tag (EST)-derived, biotin labeled antisense "capture" primers and streptavidin-coated magnetic beads. Full-length cDNA is then synthesized from purified EST-specific mRNA and cloned directly into plasmid vectors. The results of using beta-actin-based capture primers on cytoplasmic RNA were the isolation of both beta- and gamma-actin cDNA clones. Of the 16 actin-specific cDNA clones analyzed, 15 (93%) were full-length. This approach for cloning full-length cDNAs from available ESTs or partial cDNA sequences will facilitate a more rapid and efficient characterization of gene structure and function.     

Cloning and expression of cynomolgus monkey and baboon zona pellucida proteins

Partial clones for the three cynomolgus monkey (Macaca fasicularis) zona pellucida genes (cmZPA, cmZPB, and cmZPC) have previously been isolated. These partial clones contained the sequences for the C-terminal portion of each rcmZP protein. To obtain full-length clones for each cmZP, a fresh cynomolgus monkey ovarian cDNA library was constructed. PCR methodology was employed to speed the isolation of full-length clones for each cmZP cDNA. The 3' primers were designed based on sequence information from the previously identified clones; the 5' primers were designed using the human ZP sequences. The PCR technique yielded full-length clones of cmZPA and cmZPC, but not of cmZPB. Therefore, a genomic clone of cmZPB was isolated and the sequence determined. The exon/intron structure is nearly identical to the human ZPB exon/intron structure. New PCR primers were designed based on the cynomolgus monkey ZPB genomic sequence, and a full-length cmZPB cDNA was obtained. The same primers that were used to generate the cmZPB were also used to generate a baboon (Papio cynocephalus) ZPB (bZPB) cDNA. As was done previously for the human zona pellucida (hZP) cDNAs, the cmZP, and bZPB cDNAs were transferred to shuttle vectors for transfection into Chinese Hamster Ovary (CHO) cells. Stable cell lines for producing each ZP protein were isolated. Each cell line secreted the desired recombinant zona pellucida (rZP) protein into the culture medium, and each protein was purified using an established protocol. In terms of size and purity, the purified recombinant cmZP (rcmZP) and rbZPB proteins resemble the rhZP proteins.     

Regional insertional mutagenesis of genes on Arabidopsis thaliana chromosome V using the Ac/Ds transposon in combination with a cDNA scanning method

For regional insertional mutagenesis of Arabidopsis thaliana genes, we combined a cDNA scanning method (Hayashida et al. Gene 1995; 165:155-161) and an Ac/Ds transposon designed for local mutagenesis, and evaluated this approach with two overlapping yeast artificial chromosome (YAC) clones, CIC7E11 and CIC8B11, on A. thaliana chromosome 5. We applied a previously developed novel cDNA selection method using DNA latex particles (cDNA scanning method) to the two YAC clones and constructed two sub-libraries in which cDNAs for genes on each YAC DNA were concentrated. From each sub-library we isolated cDNAs for genes on each YAC DNA, partially sequenced them, and produced expressed sequence tags (ESTs). In total, 113 non-redundant groups of cDNAs were obtained. Forty-four per cent of these EST clones were novel, and 34% had significant homology to functional proteins from various organisms. In parallel, we transposed Ds from a donor Ds-GUS-T-DNA line, Ds4391-20, already mapped to the CIC7E11/8B11 region. We obtained Ds-transposed lines and recovered their Ds-flanking genomic DNAs by thermal asymmetric interlaced (TAIL) polymerase chain reaction (PCR). Dot-blot analysis indicated that 20% of the lines contained transposed Ds in the CIC7E11/8B11 region, suggesting that this Ac/Ds transposon system is effective for regional insertional mutagenesis. To isolate Ds insertion mutants in the genes identified from the CIC7E11/8B11 region, we carried out PCR screening from 800 Ds-containing lines using Ds-specific and gene-specific primers that were designed from the 113 cDNA sequences identified by the cDNA scanning method. We found that 49 lines contain Ds insertion mutations, and that five lines contain Ds mutations in genes that are mapped to the sequenced CIC7E11/8B11 genomic DNA region. These results indicate that combining the cDNA scanning method and the Ac/Ds transposon gives a powerful tool for regional insertional mutagenesis not only in Arabidopsis but also in other plants or crops whose genomes are not sequenced.     

Cloning full-length, cap-trapper-selected cDNAs by using the single-strand linker ligation method.

We have developed the single-strand linker ligation method (SSLLM), which uses DNA ligase to add a dsDNA linker to single-stranded (ss) full-length cDNA. The linkers have random 6-bp (dN6 or dGN5) 3' overhangs that can ligate to any cDNA sequence, thereby facilitating the production of cDNA libraries with titers exceeding 1 x 10(6) independent clones. We confirmed that the 5' ends of cDNA inserts cloned by using SSLLM are full-length and include the 5' untranslated regions. The great advantage of our method is that the elimination of the GC tail simplifies the sequencing and protein translation of the full-length clones. Further, our method tags ss cDNAs more efficiently than does the traditional RNA ligase reaction.     

A rapid and cost-effective method for sequencing pooled cDNA clones by using a combination of transposon insertion and Gateway technology

Large-scale cDNA-sequencing projects require an efficient strategy for mass sequencing. Here we describe a method for sequencing pooled cDNA clones using a combination of transposon insertion and Gateway technology. Our method reduces the number of shotgun clones that are unsuitable for reconstruction of cDNA sequences, and has the advantage of reducing the total costs of the sequencing project.     

Sequencing and Analysis of Approximately 40 000 Soybean cDNA Clones from a Full-Length-Enriched cDNA Library

A large collection of full-length cDNAs is essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. We obtained a total of 39 936 soybean cDNA clones (GMFL01 and GMFL02 clone sets) in a full-length-enriched cDNA library which was constructed from soybean plants that were grown under various developmental and environmental conditions. Sequencing from 5′ and 3′ ends of the clones generated 68 661 expressed sequence tags (ESTs). The EST sequences were clustered into 22 674 scaffolds involving 2580 full-length sequences. In addition, we sequenced 4712 full-length cDNAs. After removing overlaps, we obtained 6570 new full-length sequences of soybean cDNAs so far. Our data indicated that 87.7% of the soybean cDNA clones contain complete coding sequences in addition to 5′- and 3′-untranslated regions. All of the obtained data confirmed that our collection of soybean full-length cDNAs covers a wide variety of genes. Comparative analysis between the derived sequences from soybean and Arabidopsis, rice or other legumes data revealed that some specific genes were involved in our collection and a large part of them could be annotated to unknown functions. A large set of soybean full-length cDNA clones reported in this study will serve as a useful resource for gene discovery from soybean and will also aid a precise annotation of the soybean genome.Key words: EST, full-length cDNA, functional annotation, legume, soybean     

TAIL-PCR方法快速分离Xcc致病相关基因序列

以mini-Tn5 gfp-km转座子中nptⅡ片段作为探针,对已获得的五株野油菜黄单胞菌野油菜黑腐病致病型（Xcc)非致病突变体进行了Southern blot分析,结果表明,这五株突变体确由mini-Tn5 gfp-km转座子插入致病相关基因所致,且为单拷贝不同位点的插入。提取这五株突变体总DNA作为模板,采用改进的热不对称交错PCR（TAIL－PCR）方法从其中克隆到了各自转座子插入区侧翼序列,对这些侧翼序列进行了序列测定并将分析结果与GenBank database及Xcc全基因组序列做了比较,结果表明,五个侧翼序列所在的基因确与Xcc致病性有关。这种改进后的TAIL－PCR方法为突变体特别是转座子插入突变体中目的基因的克隆提供了一种简要高效的新方法。     

Direct cloning of a target gene from a pool of homologous sequences: complete cDNA sequence of a weak neurotoxin from cobra Naja kaouthia

Selective cloning of the cDNA coding for a weak neurotoxin (WTX) from cobra N. kaouthia including the 5'- and 3'-non-translated regions (NTR) is described. The known amino acid sequence of WTX was used together with the nucleotide sequence of a weak neurotoxin NNAM2 from cobra Naja atra, to design WTX-specific primers for direct amplification of an internal WTX cDNA fragment by RT- PCR. The sequence of the complete WTX cDNA was determined in sequencing runs on internal PCR products, cloned 3'- and 5'-RACE-fragments and several full-length cDNA clones. The cDNA coding sequence is in excellent agreement with the previously determined WTX amino acid sequence, has a high homology with other known weak toxin cDNAs, whereas even higher homology (up to 96%) with several classes of 3-finger toxins was detected in the 59 bp 3'-NTR consensus sequence. A possible function of the highly conserved nucleotide sequence elements is discussed.     

用基因组DNA剪接技术克隆SIgA相关基因

目的：克隆分泌型IgA（SIgA）相关基因--J链基因(IgJ)、多聚免疫球蛋白受体基因（pIgR）和IgA重链恒定区基因（IGHA）,为进一步构建SIgA真核表达质粒奠定基础。方法：采用本室建立的"基因组DNA剪接"技术,根据已发表的IgJ、pIgR和IGHA的核苷酸序列,通过计算机软件分别设计各个基因片段外显子的优化引物,从人外周血基因组DNA中直接扩增各基因的外显子序列;然后人工设计融合相邻外显子的融合引物,采用重叠PCR技术,把各基因片段的外显子串联起来形成全长编码序列,完成基因组DNA的体外剪接。扩增的PCR产物纯化后克隆到pGEM-T Easy Vector中,通过DNA测序对阳性克隆进行分析鉴定。结果：PCR扩增的IgJ、pIgR和IGHA基因与预期大小一致;测序结果表明本实验获得的上述基因与GenBank中的目标基因序列完全一致。结论：本文通过基因组DNA剪接技术成功克隆人类SIgA三个相关基因,提示此技术是合成多外显子cDNA的有效手段。     

Generation of full-length cDNA libraries enriched for differentially expressed genes for functional genomics

Here, we describe the application of a RecA-based cloning technology to generate full-length cDNA libraries enriched for genes that are differentially expressed between tumor and normal tissue samples. First, we show that the RecA-based method can be used to enrich cDNA libraries for several target genes in a single reaction. Then, we demonstrate that this method can be extended to enrich a cDNA library for many full-length cDNA clones using fragments derived from a subtracted cDNA population. The results of these studies show that this RecA-mediated cloning technology can be used to convert subtracted cDNAs or a mixture of several cDNA fragments corresponding to differentially expressed genes into a full-length library in a single reaction. This procedure yields a population of expression-ready clones that can be used for further high-throughput functional screening.     

A simple,rapid, efficient and inexpensive strategy for sequencing clones from cDNA libraries

In this report, we describe a simple, rapid, efficient and inexpensive strategy for sequencing inserted DNAs from clones of cDNA or gDNA libraries. This strategy uses PCR products directly amplified from transformed bacterial colonies, with universal primers within the vector. The method can be applied for sequencing cDNA or gDNA libraries with up to 4 ∼ 5 kb insert sizes, without overnight liquid culture or plasmid DNA preparation steps. We successfully used this method to analyze clones from full-length, enriched cDNA libraries. Although simple, following this strategy will significantly help researchers to avoid unnecessary steps in the analysis of a cDNA library.     

Novel mutants of Erwinia carotovora subsp. carotovora defective in the production of plant cell wall degrading enzymes generated by Mu transpososome-mediated insertion mutagenesis

As in Erwinia carotovora subsp. carotovora the regulation details of the main virulence factors, encoding extracellular enzymes that degrade the plant cell wall, is only rudimentally understood, we performed a genetic screen to identify novel candidate genes involved in the process. Initially, we used Mu transpososome-mediated mutagenesis approach to generate a comprehensive transposon insertion mutant library of ca. 10000 clones and screened the clones for the loss of extracellular enzyme production. Extracellular enzymes production was abolished by mutations in the chromosomal helEcc, trkAEcc yheLEcc, glsEcc, igaAEcc and cysQEcc genes. The findings reported here demonstrate that we have isolated six new representatives that belong to the pool of genes modulating the production of virulence factors in E. carotovora.     

Development of Full-Length cDNAs from Chinese Cabbage (Brassica rapa Subsp. pekinensis) and Identification of Marker Genes for Defence Response

Arabidopsis belongs to the Brassicaceae family and plays an important role as a model plant for which researchers have developed fine-tuned genome resources. Genome sequencing projects have been initiated for other members of the Brassicaceae family. Among these projects, research on Chinese cabbage (Brassica rapa subsp. pekinensis) started early because of strong interest in this species. Here, we report the development of a library of Chinese cabbage full-length cDNA clones, the RIKEN BRC B. rapa full-length cDNA (BBRAF) resource, to accelerate research on Brassica species. We sequenced 10 000 BBRAF clones and confirmed 5476 independent clones. Most of these cDNAs showed high homology to Arabidopsis genes, but we also obtained more than 200 cDNA clones that lacked any sequence homology to Arabidopsis genes. We also successfully identified several possible candidate marker genes for plant defence responses from our analysis of the expression of the Brassica counterparts of Arabidopsis marker genes in response to salicylic acid and jasmonic acid. We compared gene expression of these markers in several Chinese cabbage cultivars. Our BBRAF cDNA resource will be publicly available from the RIKEN Bioresource Center and will help researchers to transfer Arabidopsis-related knowledge to Brassica crops.     

Identification and characterization of three genes and two pseudogenes on chromosome 13

A study was conducted on the feasibility of isolating genes and pseudogenes that map to chromosome 13 by a hybridization-based approach using a 13-specific library and pools of repeat-free cDNA clones. Five pairs of cDNA and chromosome 13 genomic clones were identified and characterized. Partial or full-length sequence was derived from all cDNAs, and database searches were performed for putative gene identification. Partial sequence was also obtained from the chromosome 13 genomic clones for comparison with those of the hybridizing cDNAs. As a result of these analyses we identified three genes, a putative homologue of a porcine mRNA encoding an unidentified hepatic protein, a putative homologue of a yeast integral membrane protein, and a gene for a translationally controlled tumor protein, and two processed pseudogenes, ribosomal proteins L23a and S3a. The latter was formerly identified as the v-fos transformation effector gene, Fte-1, and recently cited as a possible candidate for the BRCA2 gene on chromosome 13. All genes and pseudogenes were localized to cytogenetic bands by in situ hybridization of metaphase chromosomes with probes derived from the chromosome 13 genomic clones.