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• W2067685979 abstract "Conditions favoring left-handed Z-DNA such as high salinity (> 4 M), 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 DH5α 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. Conditions favoring left-handed Z-DNA such as high salinity (> 4 M), 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 DH5α 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. DNA is a flexible and dynamic molecule which can adopt a variety of conformations (1Sinden R.R. DNA Structure and Function. Academic Press, San Diego, CA1994Google Scholar). Under certain conditions, some DNA sequences can change from the most prevalent right-handed B-DNA conformation and adopt unusual DNA conformations, such as left-handed Z-DNA (2Wang A.H.-J. Quigley G.J. Kolpak F.J. Crawford J.L. van Boom J.H. van der Marel G. Rich A. Nature. 1979; 282: 680-686Crossref PubMed Scopus (1579) Google Scholar), as well as triplex (3Mirkin S.M. Lyamichev V.I. Drushlyak K.N. Dobrynin V.N. Filippov S.A. Frank-Kamenetskii M.D. Nature. 1987; 330: 495-497Crossref PubMed Scopus (354) Google Scholar, 4Wells R.D. Collier D.A. Hanvey J.C. Shimizu M. Wohlrab F. FASEB J. 1988; 2: 2939-2949Crossref PubMed Scopus (491) Google Scholar), tetraplex (5Sen D. Gilbert W. Nature. 1988; 334: 364-366Crossref PubMed Scopus (1425) Google Scholar), and cruciform structures (6Panayotatos N. Wells R.D. Nature. 1981; 289: 466-470Crossref PubMed Scopus (373) Google Scholar). With increasing salt concentration, inversion of the circular dichroism spectrum of poly-d(CG) nucleotides was observed (7Pohl F.M. Jovin T.M. J. Mol. Biol. 1972; 67: 375-396Crossref PubMed Scopus (1025) Google Scholar), suggesting the occurrence of an unusual DNA form. However, detailed x-ray crystallographic analysis of d(CG) hexamer was required to established the formation of left-handed Z-DNA (2Wang A.H.-J. Quigley G.J. Kolpak F.J. Crawford J.L. van Boom J.H. van der Marel G. Rich A. Nature. 1979; 282: 680-686Crossref PubMed Scopus (1579) Google Scholar). Spectroscopic, chemical, and enzymatic analyses have subsequently been used to study the conditions required for Z-DNA formation in vitro (2Wang A.H.-J. Quigley G.J. Kolpak F.J. Crawford J.L. van Boom J.H. van der Marel G. Rich A. Nature. 1979; 282: 680-686Crossref PubMed Scopus (1579) Google Scholar, 8Johnston B.H. Methods Enzymol. 1992; 211: 127-158Crossref PubMed Scopus (34) Google Scholar, 9Palecek E. Crit. Rev. Biochem. Mol. Biol. 1991; 26: 151-226Crossref PubMed Scopus (221) Google Scholar). In our previous study (10Kim J.- Yang C.-F. DasSarma S. J. Biol. Chem. 1996; 271: 9340-9346Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), the factors required for Z-DNA formation were systematically examined by Z-DNA conformational analysis of a series of short alternating d(CG) sequences cloned in plasmids. The results showed that d(CG)4-7 sequences are in a dynamic equilibrium between B- and Z-DNA forms, while longer alternating d(CG) sequences are predominantly in the Z-form. DNA supercoiling was essential for Z-DNA formation and higher DNA supercoiling favored Z-DNA formation in shorter d(CG) repeat sequences. Surprisingly, salt concentrations as high as 4 M NaCl did not promote Z-DNA formation in these shorter alternating d(CG) sequences to a measurable extent. Naturally occurring alternating purine-pyrimidine sequences adopting Z-DNA conformation have been found previously in many organisms including prokaryotic and eukaryotic microorganisms, and plants and mammalian cells (2Wang A.H.-J. Quigley G.J. Kolpak F.J. Crawford J.L. van Boom J.H. van der Marel G. Rich A. Nature. 1979; 282: 680-686Crossref PubMed Scopus (1579) Google Scholar, 9Palecek E. Crit. Rev. Biochem. Mol. Biol. 1991; 26: 151-226Crossref PubMed Scopus (221) Google Scholar, 11Bianchi A. Wells R.D. Heintz N.H. Caddle M.S. J. Biol. Chem. 1990; 265: 21789-21796Abstract Full Text PDF PubMed Google Scholar, 12Kilpatrick M.W. Klysik J. Singleton C.K. Zarling D.A. Jovin T.M. Henau L.H. Erlanger B.F. Wells R.D. J. Biol. Chem. 1984; 259: 7268-7274Abstract Full Text PDF PubMed Google Scholar, 13Ferl R.J. Paul A.-L. Plant Mol. Biol. 1992; 18: 1181-1184Crossref PubMed Scopus (8) Google Scholar, 14Vlach J. Dvorak M. Bartunek P. Pecenka V. Travnicek M. Sponar J. Biochem. Biophys. Res. Commun. 1989; 158: 737-742Crossref PubMed Scopus (6) Google Scholar, 15Schroth G.P. Chou P.-J. Ho P.S. J. Biol. Chem. 1992; 267: 11846-11855Abstract Full Text PDF PubMed Google Scholar, 16Thomas M.J. Freeland T.M. Strobl J.S. Mol. Cell. Biol. 1990; 53: 78-87Google Scholar). The presence of Z-DNA-forming sequences in putative regulatory regions near the 5′-ends of genes (15Schroth G.P. Chou P.-J. Ho P.S. J. Biol. Chem. 1992; 267: 11846-11855Abstract Full Text PDF PubMed Google Scholar) and the inhibition of transcription by RNA polymerase in Z-DNA regions (17Peck L.J. Wang J.C. Cell. 1985; 40: 129-137Abstract Full Text PDF PubMed Scopus (103) Google Scholar) suggested a regulatory role for Z-DNA in gene expression. The finding of greater Z-DNA conformation within transcriptionally active regions of chromosomes (18Wittig B. Dorbic T. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2259-2263Crossref PubMed Scopus (119) Google Scholar) was also consistent with a role for Z-DNA in transcription. Involvement of Z-DNA in genetic recombination (19Freund A.-M. Bichara M. Fuchs R.P.P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7465-7469Crossref PubMed Scopus (76) Google Scholar, 20Blaho J.A. Wells R.D. J. Biol. Chem. 1987; 262: 6082-6088Abstract Full Text PDF PubMed Google Scholar) was supported by the finding of high frequency recombination for plasmids containing Z-DNA, preferential binding of recombinational proteins such as RecA to Z-DNA, and finding of potential Z-DNA-forming d(GT)n sequences in recombinational hot spots in eukaryotic cells. Other functions for Z-DNA, such as a structural role in nucleosome formation (21Garner M.M. Felsenfeld G. J. Mol. Biol. 1987; 196: 581-590Crossref PubMed Scopus (95) Google Scholar) and as the recognition site for DNA topoisomerase II (22Arndt-Jovin D.J. Udvardy A. Garner M.M. Ritter S. Jovin T.M. Biochemistry. 1993; 32: 4862-4872Crossref PubMed Scopus (29) Google Scholar), have also been suggested, and several Z-DNA-binding proteins have been isolated (23Zhang S. Lockshin C. Herbert A. Winter E. Rich A. EMBO J. 1992; 11: 3787-3796Crossref PubMed Scopus (203) Google Scholar, 24Herbert A.G. Spitzner J.R. Lowenhaupt K. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3339-3342Crossref PubMed Scopus (35) Google Scholar, 25Herbert A. Lowenhaupt K. Spitzner J. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7550-7554Crossref PubMed Scopus (116) Google Scholar). However, due to the dynamic nature of genetic processes and the transient nature of left-handed Z-DNA conformation, the exact roles of Z-DNA in vivo are still controversial. To gain deeper insight into the functional role of Z-DNA conformation in vivo, we explored the characteristics of a diversity of organisms and identified H. halobium as an interesting organism for the analysis of left-handed DNA conformation. H. halobium occupies an unusual evolutionary and physiological position, as a member of a group of obligately halophilic archaea (archaebacteria) requiring at least 3 M NaCl for growth. H. halobium contains high (>4 M) salt concentration (26Christian J.H.B. Waltho J.A. Biochim. Biophys. Acta. 1962; 65: 506-508Crossref PubMed Scopus (173) Google Scholar), high (σ = 0.7) negative DNA supercoiling (10Kim J.- Yang C.-F. DasSarma S. J. Biol. Chem. 1996; 271: 9340-9346Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 27Yang C.-F. Kim J.-M. Molinari E. DasSarma S. J. Bacteriol. 1996; 178: 840-845Crossref PubMed Scopus (36) Google Scholar), and high (∼67%) GC content (28Moore R.L. McCarthy B.J. J. Bacteriol. 1969; 99: 248-254Crossref PubMed Google Scholar), all of which are known to favor Z-DNA conformation. We reasoned that Z-DNA is likely to be highly prevalent in the H. halobium genome, and isolation and characterization of these sequences may provide valuable information about the function of Z-DNA sequences generally. In order to isolate Z-DNA-forming regions of the H. halobium genome, we developed an improved method for affinity chromatography using a Z-DNA-specific monoclonal antibody column (29Thomae R. Beck S. Pohl F.M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5550-5553Crossref PubMed Scopus (29) Google Scholar, 30Hoheisel J.D. Pohl F.M. J. Mol. Biol. 1987; 193: 447-464Crossref PubMed Scopus (20) Google Scholar, 31Lafer E.M. Möller A. Nordheim A. Stollar B.D. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3546-3550Crossref PubMed Scopus (196) Google Scholar). Using a plasmid series containing d(CG)n sequences, the procedure was shown to be highly selective and was used to isolate recombinant plasmids with Z-DNA-forming sequences from an H. halobium genomic library. We found that although salt has no measurable effect on Z-DNA formation in short alternating d(CG) sequences (10Kim J.- Yang C.-F. DasSarma S. J. Biol. Chem. 1996; 271: 9340-9346Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), a large number of Z-DNA-forming sequences do exist in the H. halobium genome. Restriction endonucleases, large fragment of Escherichia coli DNA polymerase I, T4 DNA ligase, S1 nuclease, and T4 polynucleotide kinase were purchased from Life Technologies, Inc. and New England Biolabs. Exonuclease III was purchased from Stratagene, and Sequenase version 2.0 DNA sequencing kit was purchased from U. S. Biochemical Corp. Affi-Gel protein A-agarose was purchased from Bio-Rad. Glutaraldehyde, OsO4, and other chemicals were purchased from Sigma. Radiolabeled nucleotides, 2′-deoxyadenosine 5′-[α-32P]triphosphate (3,000 Ci/mmol, 10 mCi/ml), and adenosine 5′-[γ-32P]triphosphate (5,000 Ci/mmol, 10 mCi/ml), were purchased from Amersham Corp. Monoclonal antibodies (Z22 and Z44) against Z-DNA were a gift of Dr. B. D. Stollar (Tufts University, Boston, MA) (31Lafer E.M. Möller A. Nordheim A. Stollar B.D. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3546-3550Crossref PubMed Scopus (196) Google Scholar) and DNA dot blots containing Halobacterium salinarum GRB cosmid library DNA (32St Jean A. Trieselmann B.A. Charlebois R.L. Nucleic Acids Res. 1994; 22: 1476-1483Crossref PubMed Scopus (14) Google Scholar) were kindly provided by Dr. R. L. Charlebois (University of Ottawa, Ontario, Canada). Standard methods of molecular biology including preparation of plasmids by the alkaline-SDS procedure, agarose and polyacrylamide gel electrophoresis, CaCl2-mediated transformation of E. coli, labeling of DNA, etc. were carried out as described (33Sambrook J. Fristch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). DNA sequencing was carried out by the dideoxy chain termination method of Sanger (34Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52251) Google Scholar) on double-stranded plasmid template using the Sequenase version 2.0 sequencing kit. Southern and dot-blot hybridizations were carried out under stringent conditions as described (33Sambrook J. Fristch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Z-DNA-containing plasmids were treated with Z22 antibody (31Lafer E.M. Möller A. Nordheim A. Stollar B.D. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3546-3550Crossref PubMed Scopus (196) Google Scholar) and isolated by affinity chromatography using protein A-agarose. Protein A affinity chromatography was prepared by packing 0.4 ml of protein A-agarose in a chromatography column. The column was equilibrated with 10 bed volumes of binding buffer (10 mM Tris-HCl (pH 8.75) and 0.1 M NaCl) before application of DNA. For control experiments, 10-µg mixtures of pUC12 and pJKCGn (n = 2, 4, and 5) (constructions described in Ref. 10Kim J.- Yang C.-F. DasSarma S. J. Biol. Chem. 1996; 271: 9340-9346Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) were incubated with 0.1 µg of Z22 antibodies in 100 µl of binding buffer at 37°C for 1 h. The mixture was passed through a protein A-agarose column prepared as above at a flow rate of 0.4 ml/min. The column was washed with 20 column volumes of binding buffer to remove nonspecifically bound DNA. The bound DNA was consecutively eluted with 2 column volumes of binding buffer with increasing ethidium bromide concentration (0.01, 0.5, 5, and 50 µg/ml). Each fraction of column volume was extracted twice with phenol and once with chloroform. The DNA was precipitated with 0.1 µg of yeast tRNA carrier, 0.1 volume of 3 M sodium acetate (pH 5.2), and 2 volumes of ethanol. DNA was collected by centrifugation, dried, and then transformed into E. coli DH5α. Transformants were plated on LB plates containing 100 µg/ml ampicillin and 40 µg/ml X-gal and incubated at 37°C overnight. For selection of Z-DNA-containing plasmids, an H. halobium genomic library, constructed by inserting partial Sau3AI fragments into the BamHI site of pUC12, was amplified (35DasSarma S. Damerval Y. Jones J.G. Tandeau de Marsac N. Mol. Microbiol. 1987; 1: 365-370Crossref PubMed Scopus (56) Google Scholar). The affinity chromatography procedure was carried out as above using 10 µg of library DNA instead of the mixture of pUC12 and pJKCGn plasmids. The column fraction corresponding to the one which showed the most enriched pJKCGn plasmid in the control experiment, was collected, and the DNA was ethanol-precipitated as described above. The DNA was transformed into E. coli DH5α for plasmid amplification. The selected plasmid population was subjected to three additional rounds of affinity selection and amplification using the same procedure. Z-DNA conformation in pJKCGn and affinity-selected plasmids were first examined by the ZIBS 1The abbreviations used are: ZIBSZ-DNA-immuno-bandshiftX-gal5-bromo-4-chloro-3-indoyl β-D-galactosidebpbase pair(s)kbkilobase pair(s). (Z-DNA-immuno-bandshift) assay using Z22 monoclonal antibody cross-linking as described (10Kim J.- Yang C.-F. DasSarma S. J. Biol. Chem. 1996; 271: 9340-9346Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Fine mapping of Z-DNA conformation on pJKZ22 was carried out either by S1 nuclease and chemical modification assay (8Johnston B.H. Methods Enzymol. 1992; 211: 127-158Crossref PubMed Scopus (34) Google Scholar, 10Kim J.- Yang C.-F. DasSarma S. J. Biol. Chem. 1996; 271: 9340-9346Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) or by a Z-DNA antibody footprinting assay (10Kim J.- Yang C.-F. DasSarma S. J. Biol. Chem. 1996; 271: 9340-9346Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). For antibody footprinting assay, plasmid DNA was cross-linked with Z22 antibody and the DNA-antibody complex was incubated in 10 µl of reaction buffer containing 50 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl2, 1 mM dithiothreitol, and 5 units of PvuII. After 3 h of incubation at 37°C, 10 µl of the above buffer containing 0.5 units of exonuclease III was added to the reaction mixture and further incubated at 37°C for 30 min. The reaction mixture was passed through a Sephadex G-10 spin column (33Sambrook J. Fristch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) and dried under vacuum. The DNA was dissolved in 16 µl of water and denatured by the addition of 4 µl of 2 N NaOH. After a 10-min incubation at room temperature, the DNA was precipitated by addition of 20 µl of 0.9 M sodium acetate and 120 µl of ethanol. The DNA was collected by centrifugation and then dissolved in 5 µl of water and then phosphorylated by addition of 5 µl of 2 × kination buffer containing 2.5 pmol of [γ-32P]ATP and 2 units of T4 polynucleotide kinase. The reaction mixture was subjected to electrophoresis on a 6% denaturing polyacrylamide gel. Z-DNA-immuno-bandshift 5-bromo-4-chloro-3-indoyl β-D-galactoside base pair(s) kilobase pair(s). For S1 nuclease assays, 1 µg of plasmid DNAs were incubated with 0.1 unit of S1 nuclease at 37°C for 10 min in 20 µl of 50 mM sodium acetate (pH 4.6), 50 mM NaCl, and 1 mM zinc acetate. The reaction mixtures were extracted twice with phenol and once with chloroform, and the DNA was precipitated with ethanol as above. The site of cleavage was mapped by primer extension using 32P-labeled forward or reverse sequencing primers and Klenow fragment of DNA polymerase I, and electrophoresis on 8% polyacrylamide, 7 M urea gels (10Kim J.- Yang C.-F. DasSarma S. J. Biol. Chem. 1996; 271: 9340-9346Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). For OsO4 modification assays, plasmid DNAs were incubated in a 50-µl solution containing 25 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 2 mM EDTA, 2 mM OsO4, and 2 mM bipyrimidine at 37°C for 10 min. The reaction mixtures were passed through a Sephadex G-50 spin column, and the DNA was precipitated with ethanol. OsO4-sensitive sites were mapped by primer extension analysis as described above. In order to isolate naturally occurring DNA sequences adopting Z-DNA conformation, a highly selective affinity chromatography method was developed. The method involved incubation of plasmid DNA with Z22 monoclonal antibody specific for Z-DNA, followed by chromatography through a protein A-agarose column. The Z-DNA-containing plasmids that were complexed to antibody were retained on the column via protein A-antibody bridges, whereas other plasmids lacking Z-DNA were eluted. Bound plasmids were eluted by increasing concentrations of ethidium bromide, which unwinds the DNA by intercalation and converts Z-DNA segments into B-DNA conformation. This led to loss of antibody binding and elution of Z-positive plasmids from the column. To determine the efficiency of the affinity chromatography to select Z-positive plasmids, control experiments using mixtures of pUC12 and pJKCGn (n = 2, 4, and 5, where n refers to the number of CG dinucleotide repeats) were carried out. Plasmid DNA mixtures were incubated with Z22 antibodies and passed through the protein A-agarose column. The column was washed with excess binding buffer to remove nonspecifically bound DNA. The bound DNA in the column was eluted using increasing concentrations of ethidium bromide. Fractions were collected, and the DNA was purified by extraction with phenol and chloroform and by ethanol precipitation. The purified DNA was used to transform E. coli DH5α, and transformants were plated on LB plates containing ampicillin and X-gal. The quantity of plasmid DNA in each fraction was determined by counting the total number of transformants. Transformants containing pUC12 produced blue colonies on plates with X-gal, whereas transformants containing pJKCG2, pJKCG4, and pJKCG5 plasmids produced white colonies, due to frameshifts in the LacZα coding region caused by the d(CG)n inserts. Therefore, enrichment by affinity purification could be easily determined by counting the number of blue and white colonies. Fig. 1 shows the plasmid elution profile using the buffer containing ethidium bromide. Not shown is that more than 90% of plasmids applied to the column were either not bound or washed off the column by buffer lacking ethidium bromide. Plasmids were quantitatively released with 0.5 µg/ml and 5 µg/ml ethidium bromide with the highest enrichment of Z-positive plasmids observed at the higher concentration. Better enrichment was observed for pJKCG5 than for pJKCG4, as would be expected from our earlier finding that the insert in 90% of the former and 20% of the latter are in the Z-form (10Kim J.- Yang C.-F. DasSarma S. J. Biol. Chem. 1996; 271: 9340-9346Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Using 10:1 mixtures of pUC12:pJKCG4 or pJKCG5, about 10-fold enrichment of pJKCG4 and 1,000-fold enrichment of pJKCG5 were observed in the peak fractions. A maximum of 4,000-fold enrichment was demonstrated using a 100:1 mixture of pUC12 and pJKCG5 (data not shown). After determining the high selectivity of our affinity chromatography procedure in control experiments using mixtures of pUC12 and pJKCGn plasmids, we used plasmid DNA amplified from an H. halobium genomic library to isolate natural sequences capable of adopting Z-DNA conformation. The genomic library had previously been constructed by inserting partial Sau3AI fragments into the BamHI site of pUC12 (35DasSarma S. Damerval Y. Jones J.G. Tandeau de Marsac N. Mol. Microbiol. 1987; 1: 365-370Crossref PubMed Scopus (56) Google Scholar). The genomic plasmid library was incubated with Z22 antibody, and the mixture was passed over a protein A-agarose column. After washing the column with an excess amount of binding buffer, Z-DNA-antibody complex bound to protein A-agarose was eluted with buffer containing ethidium bromide. The fractions corresponding to the most enriched fractions were collected and transformed into E. coli DH5α for amplification. Plasmid DNA was isolated from a culture of the total mixture of transformed cells. The plasmid population enriched for Z-positive plasmids was subjected to a second, third, and fourth cycle of Z-DNA selection by affinity chromatography to obtain plasmid mixtures increasingly enriched for Z-DNA. Plasmids isolated from each cycle of Z-DNA selection procedure were subjected to agarose gel electrophoresis to determine if specific plasmids were selected from the genomic library (Fig. 2). Library DNA before selection for Z-positive plasmids was highly complex and appeared as a smear upon electrophoresis (lane 2), whereas distinct bands appeared after successive passages through the affinity column (lanes 3-6). This confirmed that the procedure is selective for Z-DNA-containing plasmids with greater purity in successive cycles of chromatography. After four cycles of Z-DNA enrichment and amplification, plasmids were prepared in large scale and electrophoresed on a preparative agarose gel. After staining the gel with ethidium bromide, 20 distinct bands were observed. Each was purified by electroelution and transformed into E. coli DH5α for DNA amplification. Plasmid DNAs obtained from gel purified Z-positive bands were designated as pJKZn, where n refers to the identity of individual plasmids. Some bands yielded multiple Z-positive plasmids. The presence and approximate location of Z-DNA conformation in the isolated pJKZn plasmids was analyzed by ZIBS assay (10Kim J.- Yang C.-F. DasSarma S. J. Biol. Chem. 1996; 271: 9340-9346Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). For the ZIBS assay, plasmid DNAs were incubated with and cross-linked to Z22 antibody, digested with HinfI, and labeled with [α-32P]dATP by Klenow fragment of E. coli DNA polymerase I. The bands containing the Z-DNA segment were shifted-up due to antibody cross-linking and the band intensity decrease was used as the criterion for Z-DNA formation. Each isolated plasmid was subjected to the ZIBS assay and the results for several, pJKZ73, 81, 162, 173, 182, 183, 191, 192, and 201, are shown in Fig. 3. The intensity of one HinfI restriction fragment in each plasmid decreased upon antibody cross-linking (even-numbered lanes) compared to the same fragment intensity without antibody cross-linking (odd-numbered lanes). The occurrence of antibody binding reflected by a clearly shifted band was detected in 20 distinct recombinant plasmids, although some differences in the extent of bandshift probably reflecting the strength of antibody binding was observed. All of the Z-DNA-containing plasmids isolated are listed in Table I.TABLE I.Characteristics of cloned Z-DNA regions of H. halobiumZ-DNA regionsSize of cloned regionNumber of BssHIIsitesAntibody bindinga+, moderate antibody binding; ++, stronger antibody binding; +++, strongest antibody binding; ND, not determined.Cosmids hybridizedbCosmids dot-blots were obtained from Dr. R. Charlebois (32).kbZ110.52+++G18H11, G29G6Z120.551+++G6A12, G20A8Z210.62+++G12E9Z220.042+++Z324.514++G19E12Z331.02NDZ431.22+++G27D12Z514.515+++G14B2Z521.254+++G5H7, G14F11, G14B2Z532.75+++G6A12, G9G1Z612.86++Z624.57++Z634.08+++GGA12, G20A8Z831.32+Z1022.23++Z1823.54+++Z1831.22+++G6G4, G9D9, G9F11, G17H2Z1921.54+++G30H7Z2013.56+++G17H2, G27D12, G21D2a +, moderate antibody binding; ++, stronger antibody binding; +++, strongest antibody binding; ND, not determined.b Cosmids dot-blots were obtained from Dr. R. Charlebois (32St Jean A. Trieselmann B.A. Charlebois R.L. Nucleic Acids Res. 1994; 22: 1476-1483Crossref PubMed Scopus (14) Google Scholar). Open table in a new tab Further analysis of Z-DNA conformation in recombinant plasmids isolated by affinity chromatography was carried out by digestion with BssHII. Since BssHII recognizes the sequence G′CGCGC, the occurrence of short (dG-dC) repeats, the most likely Z-DNA-forming sequence, could be tested. The results of restriction digestion and agarose gel electrophoresis (data not shown, Ref. 36Kim, J.-M., 1995, Left-handed Z-DNA in Halobacterium halobium. Ph.D. thesis, University of Massachusetts, Amherst.Google Scholar) indicate that all isolated plasmids were cleaved by BssHII. Since there is no BssHII recognition sequence in the pUC12 vector portion, cleavage by BssHII indicates that all inserts contain at least one (dG-dC)3 repeat sequence (Table I). Several plasmids, such as pJKZ32, pJKZ51, and pJKZ63, contained many BssHII cleavage sites. These results indicate that the Z-positive plasmids may contain multiple Z-DNA-forming (dG-dC) repeat sequences. Since d(CG) repeats have high potential to form Z-DNA, it was of interest to analyze whether BssHII recognition sites indeed correlate to Z22 antibody binding and Z-DNA formation. For all plasmids tested, antibody-binding HinfI restriction fragments were cleaved by BssHII (data not shown, Ref. 36Kim, J.-M., 1995, Left-handed Z-DNA in Halobacterium halobium. Ph.D. thesis, University of Massachusetts, Amherst.Google Scholar). These results suggested that the Z-DNA-forming sequences isolated may contain alternating purine-pyrimidine sequences with at least one (dG-dC)3 repeat. In order to obtain more information about DNA sequences adopting Z-DNA conformation, limited DNA sequence analysis of several pJKZn plasmids was carried" @default.
• W2067685979 created "2016-06-24" @default.
• W2067685979 creator A5026657416 @default.
• W2067685979 creator A5028832053 @default.
• W2067685979 date "1996-08-01" @default.
• W2067685979 modified "2023-09-27" @default.
• W2067685979 title "Isolation and Chromosomal Distribution of Natural Z-DNA-forming Sequences in Halobacterium halobium" @default.
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