Vector-Capping: A Simple Method for Preparing a High-Quality Full-Length cDNA Library

Kato, Souichiro; Ohtoko, Kuniyo; Ohtake, Hideki; Kimura, Tomoko

doi:10.1093/dnares/12.1.53

Cited by 76 publications

(50 citation statements)

References 19 publications

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“…The full-length cDNA library of H. longicornis was constructed using the vector-capping method (Kato et al, 2005), and expressed sequence tags (EST) were prepared in our laboratory (Boldbaatar et al, 2010b). cDNA clones encoding ferritin were identified and selected from the EST database for further analysis.…”

Section: Identification and Characterization Of Hlfer Cdna Clonesmentioning

confidence: 99%

Multiple ferritins are vital to successful blood feeding and reproduction of the hard tick Haemaphysalis longicornis

Galay

Aung

Umemiya‐Shirafuji

et al. 2013

Journal of Experimental Biology

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SUMMARYTicks are obligate hematophagous parasites and important vectors of diseases. The large amount of blood they consume contains great quantities of iron, an essential but also toxic element. The function of ferritin, an iron storage protein, and iron metabolism in ticks need to be further elucidated. Here, we investigated the function a newly identified secreted ferritin from the hard tick Haemaphysalis longicornis (HlFER2), together with the previously identified intracellular ferritin (HlFER1). Recombinant ferritins, expressed in Escherichia coli, were used for anti-serum preparation and were also assayed for iron-binding activity. RT-PCR and western blot analyses of different organs and developmental stages of the tick during blood feeding were performed. The localization of ferritins in different organs was demonstrated through an indirect immunofluorescent antibody test. RNA interference (RNAi) was performed to evaluate the importance of ferritin in blood feeding and reproduction of ticks. The midgut was also examined after RNAi using light and transmission electron microscopy. RT-PCR showed differences in gene expression in some organs and developmental stages. Interestingly, only HlFER2 was detected in the ovary during oviposition and in the egg despite the low mRNA transcript. RNAi induced a reduction in post-blood meal body weight, high mortality and decreased fecundity. The expression of vitellogenin genes was affected by silencing of ferritin. Abnormalities in digestive cells, including disrupted microvilli, and alteration of digestive activity were also observed. Taken altogether, our results show that the iron storage and protective functions of ferritin are crucial to successful blood feeding and reproduction of H. longicornis. Supplementary material available online at

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Section: Identification and Characterization Of Hlfer Cdna Clonesmentioning

confidence: 99%

Multiple ferritins are vital to successful blood feeding and reproduction of the hard tick Haemaphysalis longicornis

Galay

Aung

Umemiya‐Shirafuji

et al. 2013

Journal of Experimental Biology

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“…The key to the success of a cDNA library construction are the quality of cDNA and the ability to obtain high-quality cDNAs from limited amounts of mRNA. Numerous improvements carried out in the past few years have been made in the construction of full-length cDNA libraries (Seki et al, 1998;Zhu et al, 2001;Kato et al, 2005;Ni et al, 2007). Nevertheless, the construction of high-quality cDNA libraries is still a great challenge, and there is no single method that can resolve all the common problems associated with ligation-assisted conventional cDNA library construction.…”

Section: Construction and Composition Of A Cdna Library Of O Pumilamentioning

confidence: 99%

Construction of a cDNA Library from the Ephemeral Plant Olimarabidopsis pumila and Preliminary Analysis of Expressed Sequence Tags

Zhao

Wei

Zhao

et al. 2013

Zeitschrift Für Naturforschung C

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Olimarabidopsis pumila is a close relative of the model plant Arabidopsis thaliana but, unlike A. thaliana, it is a salt-tolerant ephemeral plant that is widely distributed in semi-arid and semi-salinized regions of the Xinjiang region of China, thus providing an ideal candidate plant system for salt tolerance gene mining. A good-quality cDNA library was constructed using cap antibody to enrich full-length cDNA with the gateway technology allowing library construction without traditional methods of cloning by use of restriction enzymes. A preliminary analysis of expressed sequence tags (ESTs) was carried out. The titers of the primary and the normalized cDNA library were 1.6 · 10 6 cfu/mL and 6.7 · 10 6 cfu/mL, respectively. A total of 1093 clones were randomly selected from the normalized library for EST sequencing. By sequence analysis, 894 high-quality ESTs were generated and assembled into 736 unique sequences consisting of 72 contigs and 664 singletons. The resulting unigenes were categorized according to the gene ontology (GO) hierarchy. The potential roles of gene products associated with stress-related ESTs are discussed. The 736 unigenes were similar to A. thaliana, A. lyrata, or Thellungiella salsuginea. This research provides an overview of the mRNA expression profi le and fi rst-hand information of gene sequence expressed in young leaves of O. pumila.

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“…A stigma full-length cDNA library of the S 9 homozygote was constructed by Hitachi High-Technologies using the vector-capping method (Kato et al, 2005). An alkaliphosphatase-labeled MLPK cDNA probe was prepared using Alkphos Direct (GE Healthcare), and the stigma cDNA libraries were screened by plaque or colony hybridization.…”

Section: Rt-pcr and Racementioning

confidence: 99%

Two Distinct Forms ofM-Locus Protein Kinase Localize to the Plasma Membrane and Interact Directly withS-Locus Receptor Kinase to Transduce Self-Incompatibility Signaling inBrassica rapa

et al. 2007

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Many flowering plants possess systems of self-incompatibility (SI) to prevent inbreeding. In Brassica, SI recognition is controlled by the multiallelic gene complex (S-haplotypes) at the S-locus, which encodes both the male determinant S-locus protein 11 (SP11/SCR) and the female determinant S-receptor kinase (SRK). Upon self-pollination, the S-haplotype-specific interaction between the pollen-borne SP11 and the cognate stigmatic SRK receptor induces SI signaling in the stigmatic papilla cell and results in rejection of the self-pollen. Our genetic analysis of a self-compatible mutant revealed the involvement of a cytoplasmic protein kinase, M-locus protein kinase (MLPK), in the SI signaling, but its exact physiological function remains unknown. In this study, we identified two different MLPK transcripts, MLPKf1 and MLPKf2, which are produced using alternative transcriptional initiation sites and encode two isoforms that differ only at the N termini. While MLPKf1 and MLPKf2 exhibited distinct expression profiles, both were expressed in papilla cells. MLPKf1 localizes to the plasma membrane through its N-terminal myristoylation motif, while MLPKf2 localizes to the plasma membrane through its N-terminal hydrophobic region. Although both MLPKf1 and MLPKf2 could independently complement the mlpk/mlpk mutation, their mutant forms that lack the plasma membrane localization motifs failed to complement the mutation. Furthermore, a bimolecular fluorescence complementation assay revealed direct interactions between SRK and the MLPK isoforms in planta. These results suggest that MLPK isoforms localize to the papilla cell membrane and interact directly with SRK to transduce SI signaling.

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Vector-Capping: A Simple Method for Preparing a High-Quality Full-Length cDNA Library

Cited by 76 publications

References 19 publications

Multiple ferritins are vital to successful blood feeding and reproduction of the hard tick Haemaphysalis longicornis

Multiple ferritins are vital to successful blood feeding and reproduction of the hard tick Haemaphysalis longicornis

Construction of a cDNA Library from the Ephemeral Plant Olimarabidopsis pumila and Preliminary Analysis of Expressed Sequence Tags

Two Distinct Forms ofM-Locus Protein Kinase Localize to the Plasma Membrane and Interact Directly withS-Locus Receptor Kinase to Transduce Self-Incompatibility Signaling inBrassica rapa

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