High-Efficiency Thermal Asymmetric Interlaced PCR for Amplification of Unknown Flanking Sequences

Liu, Yao-Guang; Chen, Yuanling

doi:10.2144/000112601

Cited by 512 publications

(394 citation statements)

References 14 publications

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“…However, in some other lines, only some of these segments were amplified (Figure 1A-1P), indicating the presence of S352c-related structure variants. From these lines we then amplified and sequenced the unknown sequences flanking these known sequences using hiTAIL-PCR [13]. As a result, we identified eleven mitochondrial recombinant structures (mitotypes) related to S352c, which contain WA352c-related putative ORFs and the same or different upstream and downstream flanking sequences ( Table 1, Figure 1).…”

Section: Identification Of Cms-related Recombinant Structuresmentioning

confidence: 99%

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Multi-step formation, evolution, and functionalization of new cytoplasmic male sterility genes in the plant mitochondrial genomes

Tang

Zheng

et al. 2016

Cell Res

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New gene origination is a major source of genomic innovations that confer phenotypic changes and biological diversity. Generation of new mitochondrial genes in plants may cause cytoplasmic male sterility (CMS), which can promote outcrossing and increase fitness. However, how mitochondrial genes originate and evolve in structure and function remains unclear. The rice Wild Abortive type of CMS is conferred by the mitochondrial gene WA352c (previously named WA352) and has been widely exploited in hybrid rice breeding. Here, we reconstruct the evolutionary trajectory of WA352c by the identification and analyses of 11 mitochondrial genomic recombinant structures related to WA352c in wild and cultivated rice. We deduce that these structures arose through multiple rearrangements among conserved mitochondrial sequences in the mitochondrial genome of the wild rice Oryza rufipogon, coupled with substoichiometric shifting and sequence variation. We identify two expressed but nonfunctional protogenes among these structures, and show that they could evolve into functional CMS genes via sequence variations that could relieve the self-inhibitory potential of the proteins. These sequence changes would endow the proteins the ability to interact with the nucleus-encoded mitochondrial protein COX11, resulting in premature programmed cell death in the anther tapetum and male sterility. Furthermore, we show that the sequences that encode the COX11-interaction domains in these WA352c-related genes have experienced purifying selection during evolution. We propose a model for the formation and evolution of new CMS genes via a "multi-recombination/protogene formation/functionalization" mechanism involving gradual variations in the structure, sequence, copy number, and function.

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Section: Identification Of Cms-related Recombinant Structuresmentioning

confidence: 99%

“…The sequences of S352a/b, S367, S356, S314a, S310, S276, S284a, and S284b were isolated by long PCR or hiTAIL-PCR [13] with specific primers (Supplementary information, Tables S1 and S2). The amplified fragments were recovered, cloned and sequenced.…”

Section: Pcr and Hitail-pcrmentioning

confidence: 99%

Multi-step formation, evolution, and functionalization of new cytoplasmic male sterility genes in the plant mitochondrial genomes

Tang

Zheng

et al. 2016

Cell Res

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“…The both flanking sequences of Tn5 were amplified and sequenced, and the ORF of zmsO was determined following the methods described previously (Cheng et al 2013;Liu and Chen 2007).…”

Section: Transposon Mutagenesis and In-frame Deletion Of Zmsomentioning

confidence: 99%

“…Among these mutants, EM236 showed a significantly decreased antimicrobial activity compared to wild type strain EC1. The inserted gene was sequenced by using the hi-TAIL PCR method (Liu and Chen 2007), and designated as zmsO, which encodes a protein with 239 amino acids and sharing high identity to the phosphopantetheinyl transferase component of siderophore synthetase (Accession number WP_022632844.1) from Dickeya solani. Domain analysis showed that ZmsO contains a conserved domain of the ACPS 4′-phosphopantetheinyl transferase superfamily in the region from the 114th to 227th amino acid, suggesting that ZmsO is likely to be a 4′-phosphopantetheinyl transferase and plays a role in post-translational modification of the enzymes involved in zeamines biosynthesis.…”

Section: Identification Of a Transposon Mutant With A Decreased Antimmentioning

confidence: 99%

A Sfp-type phosphopantetheinyl transferase ZmsO is essential for zeamines production and the virulence of Dickeya zeae

et al. 2016

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Zeamines are family of potent antibiotics and virulence determinants produced by the rice foot rot bacterial pathogen Dickeya zeae. So zeamines are important for the pathogenesis of D. zeae and development of new strategies against this devastating disease. In this study, we show that production of zeamines is positively modulated by ZmsO, which is a conserved Sfp-type phosphopantetheinyl thransferase (PPTase) associated with post-translational activation of fatty acid synthases and polyketide synthases. Deletion of zmsO significantly abolished zeamines production and attenuated the virulence of D. zeae without affect the growth rate. The zmsO gene is located at the upstream of zmsA and zmsK in the same gene cluster. Consistent with its role in post-translational modification, deletion of zmsO did not affect the transcriptional expression of zmsA and zmsK. In trans expression of the pcpS gene from Pseudomonas aeruginosa, which encodes a Sfp-type PPTase, in the zmsO deletion mutant could also fully restore the zeamines production and bacterial virulence, establishing the important role of Sfp-type PPTase in the zeamines biosynthesis and pathogenicity of D. zeae.

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“…그러나 이러한 TAIL-PCR 은 획득할 수 있는 산물의 크기가 0.3-4kb 정도로 제한적이며, 그 성공률도 20-40% 이다 (Liu and Whittier, 1995). 이러한 효 율의 제약을 극복하기 위하여 primer의 길이를 조절하는 high-efficiency TAIL PCR (Liu and Chen, 2007;Michiels et al, 2003) (Antal et al, 2004) 및 autosegment (Mukai and Nakagawa, 1996), touch-up PCR (Ailenberg and Silverman, 2000 …”

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Development of an Effective PCR Technique for Analyzing T-DNA Integration Sites in Brassica Species and Its Application

Lee¹,

Yu²,

Park

2015

HST

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Insertional mutagenesis induced by T-DNA or transposon tagging offers possibilities for analysis of gene function. However, its potential remains limited unless good methods for detecting the target locus are developed. We describe a PCR technique for efficient identification of DNA sequences adjacent to the inserted T-DNA in a higher plant, Chinese cabbage (Brassica rapa ssp. pekinensis). This strategy, which we named variable argument thermal asymmetric interlaced PCR (VA-TAIL PCR), was designed by modifying a single-step annealing-extension PCR by including a touch-up PCR protocol and using long gene-specific primers. Amplification efficiency of this PCR program was significantly increased by employing an autosegment extension method and linked sequence strategy in nested long gene-specific primers. For this technique, arbitrary degenerate (AD) primers specific to B. rapa were designed by analyzing the Integr8 proteome database. These primers showed higher accuracy and utility in the identification of flanking DNA sequences from individual transgenic Chinese cabbages in a large T-DNA inserted population. The VA-TAIL PCR method described in this study allows the identification of DNA regions flanking known DNA fragments. This method has potential biotechnological applications, being highly suitable for identification of target genomic loci in insertional mutagenesis screens.

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High-Efficiency Thermal Asymmetric Interlaced PCR for Amplification of Unknown Flanking Sequences

Cited by 512 publications

References 14 publications

Multi-step formation, evolution, and functionalization of new cytoplasmic male sterility genes in the plant mitochondrial genomes

Multi-step formation, evolution, and functionalization of new cytoplasmic male sterility genes in the plant mitochondrial genomes

A Sfp-type phosphopantetheinyl transferase ZmsO is essential for zeamines production and the virulence of Dickeya zeae

Development of an Effective PCR Technique for Analyzing T-DNA Integration Sites in Brassica Species and Its Application

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