DNA sequence and shuttle vector construction of plasmid pGL3 from Plectonema boryanum PCC 6306.
DNA sequence and shuttle vector construction of plasmid pGL3 from Plectonema boryanum PCC 6306.
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来自 Plectonema boryanum PCC 6306 的质粒 pGL3 的 DNA 序列和穿梭载体构建。
DOI：
10.1093/nar/21.3.746
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发表时间：
1993
影响因子：
14.9
通讯作者：
A. G. Atherly
中科院分区：
文献类型：
--
作者：
D. K. Walton;S. Gendel;A. G. Atherly
蓝藻是一种古老的革兰氏阴性原核生物。除了光合作用，蓝藻还具有许多其他有趣的特征：a)固氮，b)细胞分化，c)调节基因组重排（1,2）和d)内含子(3)。丝状蓝藻中这些特征的研究由于缺乏穿梭载体而受到阻碍。目前，只有一个系列的载体可用，基于Nostoc sp. PCC 7524质粒pdl(4)。许多丝状蓝藻拥有质粒，但都是隐质的。质粒的鉴定仅限于限制性基因定位和交叉杂交。一个例外是pdl(5)的复制区域及其序列(6)的隔离。本研究对boryanum Plectonema(2)的小质粒pGL3进行了测序，并构建了在Plectonema和Anabaena PCC 7120中复制的穿梭载体。质粒pGL3具有一个大的ORF，据推测参与了质粒的复制。pGL3与GenBank （version 66）和pdl(6)的比较检索（TFASTA和FASTA(7)）没有发现相似之处。质粒pGL3全长1,504 bp，包含一个大的开放阅读框（482 -1489），编码一个推测为37661 Da的蛋白。编号的起始站点从唯一的Hpal站点开始。pGL3的G + C含量为46.9%。这在Plectonema属的预期范围内，其染色体G+C含量为42- 67%(8)。ORF之后是一个双对称区域（43-58），它可能是一个依赖于rho的终止子。三个反向重复序列（IR1， IR2和IR3）分别位于ORF外的79-112,237-259和567-603位置。将pGL3在pBluescript II SK+ (Stratagene)独特的Hpal位点上进行限制性修饰，并连接到pBluescript II SK+ (Stratagene)的MCS的小位点上，构建了4.6 kb的穿梭载体pPBH201，用BssBR从pBluescript II SK+中切除整个pGL3质粒，并将其连接到MCS的两侧，用SI核酸酶处理，克隆到ppboc的EcoKV位点上。pBOC是本研究中由EcoRV完全消化pRLl(4)获得的3 kbp质粒，含有氯霉素耐药基因，是迁移（出生）位点和ColEl ori的基础。蓝藻细丝通过超声减少到一到四个细胞，准备接合。按照Thiel和Wolk(9)的方法，将穿梭载体pPBH201偶联到Plectonema和Anabaena。在BG-11培养基（10）培养皿（25 /xg/ml Cm）上进行筛选。在BG-11液体培养基（25吉格/ml Cm）中培养的细胞进行穿梭载体质粒分离（11）。穿梭载体在两种蓝藻中稳定维持。pPBH201的小体积、两个多克隆位点以及在Plectonema和Anabaena中的复制能力使其在分子遗传学分析中非常有用。此外，它与pdl复制区没有相似性的事实表明，它可以与基于pdl的穿梭载体共存。
The cyanobacteria are an ancient group of Gram-negative prokaryotes. In addition to photosynthesis, the cyanobacteria possess many other interesting features: a) nitrogen-fixation, b) cellular differentiation, c) regulated genomic rearrangements (1, 2) and, d) introns (3). Studies of these features in the filamentous cyanobacteria are hampered by a lack of shuttle vectors. Currently, there is only one series of vectors available, based on the Nostoc sp. PCC 7524 plasmid pDUl (4). Many of the filamentous cyanobacteria possess plasmids but all remain cryptic. Characterization of the plasmids has been limited to restriction mapping and cross hybridizations. An exception to this is the isolation of the replication region of pDUl (5) and its sequence (6). In this study the small plasmid pGL3 from Plectonema boryanum (2) has been sequenced and a shuttle vector which replicates in Plectonema and Anabaena PCC 7120 constructed. Plasmid pGL3 possesses one large ORF which is putatively involved in the replication of the plasmid. Comparative searches (TFASTA and FASTA (7)) of pGL3 with GenBank (version 66) and pDUl (6) revealed no similarities. Plasmid pGL3 is 1,504 bp in length and contains one large open reading frame (482 -1489) coding for a putative protein of 37661 Da. The start site for numbering begins at the unique Hpal site. The G + C content of pGL3 is 46.9 percent. This is within the expected range for the Plectonema genus which has a chromosomal G+C content of 42-67 percent (8). The ORF is followed by a region of dyad symmetry (43—58) which may be a rho dependent terminator. Three inverted repeats (IR1, IR2 and IR3) lie outside of the ORF at positions 79-112, 237-259 and 567-603, respectively. A 4.6 kb shuttle vector (pPBH201) was constructed by restriction of pGL3 at the unique Hpal site and ligation into the Smal site in the MCS of pBluescript II SK+ (Stratagene), The entire pGL3 plasmid, flanked by the MCS, was excised from pBluescript II SK+ with BssBR, treated with SI nuclease, and cloned into the EcoKV site of pBOC. pBOC is a three kbp plasmid derived in this study by the complete digestion of pRLl (4) with EcoRV, and contains the chloramphenicol resistance gene, a basis of mobility (bom) site and the ColEl ori. Cyanobacterial filaments were reduced by sonication to one to four cells in preparation for conjugation. Conjugations of shuttle vector pPBH201 into Plectonema and Anabaena were performed according to Thiel and Wolk (9). Selection was on BG-11 medium (10) petri plates (25 /xg/ml Cm). Shuttle vector plasmid isolations (11) were performed on cells grown in liquid BG-11 medium (25 jig/ml Cm). The shuttle vector was stably maintained in both cyanobacteria. The small size of pPBH201, its two multiple cloning sites, and its ability to replicate in both Plectonema and Anabaena make it very useful for molecular genetic analysis. Also, the fact that it has no similarity to the pDUl replication region suggests that it can coexist with pDUl based shuttle vectors.