Wallace Margaret M, Miller David W, Raps Shirley
Department of Biological Sciences, Hunter College, CUNY, New York, NY 10021, USA.
Arch Microbiol. 2002 Apr;177(4):332-8. doi: 10.1007/s00203-002-0397-3. Epub 2002 Feb 16.
The characterization of pMa025, a plasmid isolated from the unicellular, toxin-producing cyanobacterium Microcystis aeruginosa UV025, is described. A recombinant plasmid, pMaL [pMa025-pBluescript II SK(-)] was constructed for mapping, sequencing, and development of shuttle vectors capable of transforming both Escherichia coli and M. aeruginosa. pMa025 is 8,018 bp in length and has a G+C content of 62.3 mol%. Nineteen presumptive ORFs, ORF A - ORF S were identified using ATG or GTG as initiation codons. Fifteen different ORFs, ORF a - ORF o were identified using TGA as a degenerate codon for tryptophan. GTG was the start codon in two-thirds of the putative ORFs when TGA was the termination codon. GTG was the start codon in one-third of the putative ORFs when TGA was used as a codon for tryptophan. The deduced amino acid sequence from ORF j (3,114 bp) was significantly similar to that of a putative plasmid replication protein, RepA, from plasmid pUH24 of Synecho coccus sp. strain PCC7942. M. aeruginosa UV027 and E. coli were transformed to carbenicillin resistance with pMaL-D7, a 6.4-kb hybrid plasmid (3.46 kb pMa025, 2.95 kb pBluescript II) generated from the nested deletion strategy. pMaL-D7 will be used as a shuttle vector.
本文描述了从单细胞产毒素蓝藻铜绿微囊藻UV025中分离得到的质粒pMa025的特征。构建了重组质粒pMaL [pMa025-pBluescript II SK(-)]，用于绘制图谱、测序以及开发能够转化大肠杆菌和铜绿微囊藻的穿梭载体。pMa025长度为8018 bp，G+C含量为62.3 mol%。使用ATG或GTG作为起始密码子，鉴定出19个推定的开放阅读框，即ORF A - ORF S。使用TGA作为色氨酸的简并密码子，鉴定出15个不同的开放阅读框，即ORF a - ORF o。当TGA为终止密码子时，三分之二的推定开放阅读框以GTG作为起始密码子。当TGA用作色氨酸密码子时，三分之一的推定开放阅读框以GTG作为起始密码子。从ORF j（3114 bp）推导的氨基酸序列与来自集胞藻属菌株PCC7942的质粒pUH24的推定质粒复制蛋白RepA的序列显著相似。用通过嵌套缺失策略产生的6.4 kb杂交质粒pMaL-D7（3.46 kb pMa025，2.95 kb pBluescript II）将铜绿微囊藻UV027和大肠杆菌转化为对羧苄青霉素具有抗性。pMaL-D7将用作穿梭载体。
