Isolation and chromosomal distribution of natural Z-DNA-forming sequences in Halobacterium halobium.
Conditions favoring left-handed Z-DNA such as high salinity (> 4 ), high negative DNA supercoiling, and GC-rich DNA [statistically favoring d(CG)n repeat sequences], are all found in the extremely halophilic archaeum (archaebacterium) Halobacterium halobium. In order to identify and study Z-DNA regions of the H. halobium genome, an affinity chromatography method with high Z-DNA selection efficiency was developed. Supercoiled plasmids were incubated with a Z-DNA-specific antibody (Z22) and passed over a protein A-agarose column, and the bound plasmids were eluted using an ethidium bromide gradient. In control experiments using mixtures of pUC12 (Z-negative) and a d(CG)5-containing (Z-positive) pUC12 derivative, up to 4,000-fold enrichment of the Z-DNA-containing plasmid was demonstrated per cycle of the Z-DNA selection procedure. The selection efficiency was determined by transformation of Escherichia coli DH5alpha with eluted plasmids and blue-white screening on X-gal plates. Twenty recombinant plasmids containing Z-DNA-forming sequences of H. halobium were isolated from a genomic library using affinity chromatography. Z-DNA-forming sequences in selected plasmids were identified by bandshift and antibody footprinting assays using Z22 monoclonal antibody. Alternating purine-pyrimidine sequences ranging from 8 base pairs (bp) to 13 bp with at least a 6-bp alternating d(GC) stretch were found in the Z22 antibody binding regions of isolated plasmids. The distribution of Z-DNA-forming sequences in the Halobacterium salinarum GRB chromosome was analyzed by dot-blot hybridization of an ordered cosmid library using the cloned H. halobium Z-DNA segments as probe. Among the 11 Z-DNA segments tested, five were found to be clustered in a 100-kilobase pair region of the genome, whereas six others were distributed throughout the rest of the genome.
Kim
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Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003, USA.
DasSarma
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D001483:Base Sequence; D002846:Chromatography, Affinity; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial / Q000648:ultrastructure*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry* / Q000648:ultrastructure; D015698:Genomic Library; D019617:Halobacterium salinarum / Q000235:genetics* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids
D004269:DNA, Bacterial; D004247:DNA