Note A self-ligation method for PCR-sequencing the telomeres of Streptomyces and Mycobacterium linear replicons Yun Fan a , Yumei Dai a , Qiuxiang Cheng a , Guangjun Zhang b , Dongshu Zhang b , Ping Fang b, ⁎, Hang Wu c , Linquan Bai c, ⁎, Zixin Deng c , Zhongjun Qin a, ⁎ a Key laboratory of Synthetic Biology, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, People's Republic of China b College of Environmental Sciences & Engineering, UNEP-Tongji Institute of Environment for Sustainable Development, Tongji University, Shanghai, 200092, People's Republic of China c State Key Laboratory of Microbial Metabolism and College of Life Sciences and Biotechnology, Shanghai Jiaotong University, Shanghai, 200030, People's Republic of China abstract article info Article history: Received 2 March 2012 Received in revised form 16 April 2012 Accepted 17 April 2012 Available online 26 April 2012 Keywords: Streptomyces Mycobacterium Telomere Cloning method Actinomycete species from many genera often harbor linear plasmids and some contain linear chromosomes. A self-ligation and PCR-sequencing method was developed for identifying three novel telomere sequences of linear plasmids of Streptomyces and Mycobacterium. This and four previously described methods for actino- mycetes telomere cloning and sequencing are discussed. © 2012 Elsevier B.V. All rights reserved. Actinomycetes are a large group (more than 160 genera, including antibiotic-producers for example Streptomyces, and pathogens such as Mycobacterium) of the high G+C branch of the Gram-positive bacteria (Garrity et al., 2005; Hopwood, 2006). In contrast to most bacteria, members of some actinomycetes genera (e.g. Streptomyces and Rhodococcus) contain linear chromosomes (Hopwood, 2006; McLeod et al., 2006), while species from even more genera (e.g. Strep- tomyces, Rhodococcus, Mycobacterium, Planobispora, Actinoplanes, Micrococcus and Arthrobacter) often harbor linear plasmids (Chater and Kinashi, 2007; McLeod et al., 2006; Picardeau and Vincent, 1998). Linear plasmids range in size from 12 kb to 1800 kb; their “telomeres” contain inverted repeat sequences from 12 bp to 95 kb in length (Chater and Kinashi, 2007), and the 5′ telomeric ends are linked covalently to terminal proteins (TP: Bao and Cohen, 2001). Most Streptomyces linear plasmids and linear chromosomes have conserved telomeric palindromes which the 13-bp sequences of telomeric termini are identical (5′-CCCGCGGAGCGGG-3′) (e.g., the plasmids pSLA2 and SLP2 and the Streptomyces coelicolor and Streptomyces lividans chromosomes: Huang et al., 1998) while some contain novel telomeres which also contain multiple short palin- dromes but no conserved telomeric termini sequence (e.g., SCP1, pSHK1 and the Streptomyces griseus chromosome: Goshi et al., 2002; Guo et al., 2011; Kinashi et al., 1991). Four methods for cloning the telomeres of actinomycete linear chromosomes and linear plasmids were previously reported (Fig. 1). The first Streptomyces telomere was cloned by a strategy of “force- cloning” (Hirochika et al., 1984). TP can be removed completely from its covalently-linked DNA by alkali treatment, releasing blunt ends with 5′-phosphate and 3′-hydroxy termini. However, proteinase treatment of the TP-DNA sometimes leaves several amino acids bond- ed to the DNA, preventing telomeric termini from being cloned (Goshi et al., 2002). Glass beads preferentially bind protein–DNA complexes rather than DNA alone, which is used to enrich telomere fragments of Streptomyces linear chromosomes and linear plasmids before force-cloning (Huang et al., 1998). To clone the telomere- specific segment, an Escherichia coli vector was integrated into the Rhodococcus chromosome at the telomere sequence via homologous recombination. The integrated vector along with the telomere was re- leased by digestion with a restriction enzyme which cuts within the vector to leave one blunt end (McLeod et al., 2006). To locate and PCR-sequence telomeric termini without cloning in E. coli, adenine- homopolymer [poly(A)] tails are attached to the naked 3′ ends of the Streptomyces linear chromosome by treatment with terminal deoxynucleotidyl transferase and PCR twice (nested) with one primer containing the poly(T) tails and other primers near the telomeric ter- minus (Ohnishi et al., 2008). To avoid laborious cloning in E. coli (the methods 1-3) and possi- ble artifact in the nested PCR (method 4), here we report the design of Journal of Microbiological Methods 90 (2012) 105–107 ⁎ Corresponding authors at: Shanghai Institute of Plant Physiology and Ecology, 300 Fenglin Road, Shanghai 200032, China. Tel.: + 86 21 54924171; fax: + 86 21 54924176. E-mail addresses: fangpingqin@yahoo.com.cn (P. Fang), bailq@sjtu.edu.cn (L. Bai), qin@sibs.ac.cn (Z. Qin). 0167-7012/$ – see front matter © 2012 Elsevier B.V. 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