Cloning and DNA sequence analysis of a plasmid from Helicobacter pylori




Minnis, Jennifer

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Helicobacter pylori is a pathogenic bacterium that resides in the gastric epithelium of some individuals and is widely regarded as a cause of type B gastritis and duodenal ulcer. Many bacterial species contain plasmid DNA which encodes virulence factors. Little is known about the plasmid DNAs found in some isolates of H. pylori. The purpose of this study was to clone a plasmid from Helicobacter pylori and to determine and analyze the DNA sequence.

A 3.5 kb plasmid (pHPM180) was isolated from a strain of H. pylori (HPM180) obtained from a patient with inactive duodenal ulcer. The plasmid was successfully cloned in both orientations in E. coli DH5αF\sp using the M13mp18 vector, and the DNA sequence was determined using the dideoxy chain termination method. Analysis of the sequence was performed with DNAsis and PC-Gene software packages. Two open reading frames (ORFs) were identified. ORF1 translated to 463 amino acids and a putative polypeptide with a molecular weight of 54517. ORF2 translated to 240 amino acids with a molecular weight of 28142. Ribosome binding consensus sequences were identified upstream from both ORFs, and promoter consensus sequences were identified upstream from ORF1. A 232 base pair direct repeat was also identified in the DNA sequence of pHPM180. Extensive DNA sequence homology was found between pHPM180 ORF1 and a 684 base pair HindIII fragment of a 7.2 kb H. pylori plasmid. Additionally, sequence identity was found between 142 bases in a 200 base pair overlap of pHPM180 DNA and a segment of H. pylori pHPK255; a plasmid that encodes a Gram-positive type replication protein. However, the area of homology was not contained within the ORF of pHPK255. A ribonuclease protection assay determined that ORF1 is transcribed in H. pylori HPM180.



Biological Sciences, Microbiology, Molecular biology