Off-campus UMass Amherst users: To download dissertations, please use the following link to log into our proxy server with your UMass Amherst user name and password.
Non-UMass Amherst users, please click the view more button below to purchase a copy of this dissertation from Proquest.
(Some titles may also be available free of charge in our Open Access Dissertation Collection, so please check there first.)
Analysis of the gas vesicle plasmidpNRC100 from Halobacterium halobium: Characterization of inversions, deletions, and the origin of replication
Abstract
Halobacterium halobium strain NRC-1 harbors a 200-kilobase pair (kb) plasmid, pNRC100, which contains a cluster of genes that code for synthesis of buoyant gas-filled vesicles. A restriction map of pNRC100 was constructed using AflII, DraI, HindIII, and SfiI with the aid of pulsed-field agarose gel electrophoresis. The identity and approximate positions of seventeen insertion sequences (IS) in pNRC100 were determined by Southern hybridization and limited nucleotide sequence analysis across the IS element-target site junctions: ISH2, a 0.5-kb element, was found in four copies; ISH3, a 1.4-kb heterogeneous family of elements, was present in seven copies; ISH8, a 1.4-kb element, was found in five copies; and ISH50, a 1.0-kb element, was present in a single copy. Additionally, a large (35-38 kb) inverted repeat (IR) sequence was also found. Inversion isomers of pNRC100 were demonstrated by Southern hybridization analysis using two restriction enzymes, AflII and SfiI, that cut asymmetrically within the intervening small single copy (SSC) region and the large single copy (LSC) region, respectively, but not within the large IRs. No inversion isomers were observed for a deletion derivative of pNRC100 lacking one IR, which suggests that both copies are required for inversion to occur. One end of the large IRs terminated at an ISH2 element while the other end terminated at an ISH3 element. The pNRC100 derivatives had suffered deletions which removed the gas vesicle genes and flanking DNA sequences. An autonomous replicating sequence of pNRC100 was mapped to the 19-kb HindIII-C fragment by cloning it into an E. coli plasmid containing the Haloferax volcanii mevinolin-resistance (mev$\sp{R})$ gene, transformation of H. halobium with the resulting plasmid pNGHCMEV1, and recovery of pNGHCMEV1 DNA from mevinolin-resistance transformants (Rep$\sp+).$ The nucleotide sequence of the replication origin was determined and found to contain a 550 bp highly A+T-rich segment and a 3,027 nucleotide open reading frame, named repH. The repH transcription start site was shown by primer extension analysis to be 17-18 nucleotides upstream of the putative start codon. The predicted RepH protein, an acidic protein of 113,442 molecular weight, showed 24.2 to 27.1% identity with the putative gene products of Hf. volcanii plasmid pHV2 and H. halobium halophage ($\phi$H)-derived plasmid p$\phi$HL. The replication origin was subjected to linker scanning mutagenesis and the results showed that repH is required for replication. (Abstract shortened by UMI.)
Subject Area
Microbiology|Genetics
Recommended Citation
Ng, Wai-Lap, "Analysis of the gas vesicle plasmidpNRC100 from Halobacterium halobium: Characterization of inversions, deletions, and the origin of replication" (1993). Doctoral Dissertations Available from Proquest. AAI9329650.
https://scholarworks.umass.edu/dissertations/AAI9329650