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1.
Anal Biochem ; 185(2): 230-4, 1990 Mar.
Article in English | MEDLINE | ID: mdl-2160201

ABSTRACT

A method for the efficient preparation of high quality bacteriophage lambda DNA from cleared lysates is described. Advantages of the method include high DNA yields (typically around 0.8 micrograms of DNA/1 ml of cleared lysate), speed of processing (approximately 2 h from lysate to DNA), economy, and the absence of any requirement for phenol or chloroform extractions. The technique involves the concentration of phage particles by standard polyethylene glycol precipitation followed by enzymatic treatment to remove contaminating RNA and DNA. Phage particles are then lysed with sodium dodecyl sulfate (SDS) at elevated pH and temperature. Contaminating protein/SDS complexes are rendered insoluble by the addition of potassium acetate and removed by centrifugation. The quality of the resultant DNA is comparable to that prepared by cesium chloride banding for all standard molecular biological purposes providing that spermidine is included in all restriction endonucleases digestions.


Subject(s)
Bacteriophage lambda/analysis , DNA, Viral/isolation & purification , Acetates , Acetic Acid , Chemical Precipitation , Endonucleases/metabolism , Hydrogen-Ion Concentration , Methods , Polyethylene Glycols , Sodium Dodecyl Sulfate , Temperature
2.
Anal Biochem ; 181(2): 227-33, 1989 Sep.
Article in English | MEDLINE | ID: mdl-2530918

ABSTRACT

The newly designed equipment for alternating field gel electrophoresis which permits the separation of very large DNA molecules and the simultaneous analysis of up to 35 samples is described. The field alternation is effected by intermittently rotating the submerged agarose gel by optitional angles. The time intervals between changes of position are controlled by a computer program driving a simple switching device which was designed to suit any technique using periodic switching or inversion of the electrical field. Because the electrophoresis unit provides an absolutely homogeneous electrical field, no distortion of migration lanes occurs and the resolution is very good. Moreover, by using a switching time interval gradient an almost perfect linear relationship between migration distances and molecule sizes in the range of about 100-1250 kilobase pairs is observed. In two-dimensional separations, different switching time programs for the first and second dimension allow maximum resolution of selected size ranges. Field inversion gel electrophoresis is possible as well. The performance of the method is demonstrated by comparing the chromosome sizes of different yeast strains.


Subject(s)
DNA/isolation & purification , Electrophoresis, Agar Gel/instrumentation , Bacteriophage lambda/analysis , Chromosomes, Fungal , Computers , DNA, Viral/analysis , Electromagnetic Fields , Electrophoresis , Ethidium , Indicators and Reagents , Temperature , Yeasts/analysis
3.
Anal Biochem ; 181(2): 197-208, 1989 Sep.
Article in English | MEDLINE | ID: mdl-2554760

ABSTRACT

Techniques for analyzing DNA distributions on agarose gels are examined by both two-dimensional and one-dimensional methods. It is demonstrated that very large errors in DNA concentration occur in such analyses unless (i) the electrophoresis is performed in a careful, reproducible manner, (ii) the films are calibrated with an internal standard, (iii) high resolution densitometry is used for analyzing the films, and (iv) appropriate background controls are used to determine the baselines for integration. Two-dimensional scanning produces more accurate results than one-dimensional scanning, but in cases where the bands are relatively uniform, the one-dimensional analysis gives good results. A technique for determining accurate distributions is described.


Subject(s)
DNA, Viral/analysis , Ethidium , Intercalating Agents , Bacteriophage lambda/analysis , Bacteriophages/analysis , DNA Restriction Enzymes , Densitometry , Electrophoresis, Agar Gel , Molecular Weight , Spectrometry, Fluorescence
4.
Biochemistry ; 28(14): 5827-32, 1989 Jul 11.
Article in English | MEDLINE | ID: mdl-2528375

ABSTRACT

Pulsed field agarose gel (PFG) electrophoresis, originally used to improve the resolution by length of linear DNA [Cantor et al. (1988) Annu. Rev. Biophys. Biophys. Chem. 17, 287-304], is found here to cause atypical sieving of 48.5-97.0-kb open circular DNA. Two procedures of PFG electrophoresis are used: rotating gel electrophoresis with rotation of 2 pi radians [2 pi RGE; Serwer, P., & Hayes, S.J. (1989) Appl. Theor. Electrophor. (in press)] and field inversion gel electrophoresis [FIGE; Carle, G.F., Frank, M., & Olson, M. V. (1986) Science 232, 65-68]. During 2 pi RGE at 6 V/cm, the electrophoretic mobility (mu) of 48.5-kb open circular DNA increases in magnitude as agarose percentage (A) increases from 0.4 to 1.5. The sieving revealed by this mu vs A relationship is highly atypical (possibly unique) for any particle. The extent of this atypical sieving increases as electrical potential gradient, DNA length, and pulse time increase. In some cases a maximum is observed in a plot of mu's magnitude vs A. The mu of open circular lambda DNA is smaller in magnitude than the mu of equally long linear lambda DNA. Atypical sieving has also been observed by use of FIGE. As pulse times used during FIGE decrease below those achievable by 2 pi RGE, the progressive loss of circular DNA's atypical sieving is accompanied by both a dramatic increase in mu's magnitude at the lower A values and a decrease in mu's magnitude at the higher A values. At the lower A values, open circular DNA sometimes migrates more rapidly than linear DNA of the same length.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
DNA, Circular/isolation & purification , Bacteriophage lambda/analysis , DNA, Viral/isolation & purification , Electrophoresis, Agar Gel , Models, Theoretical
6.
Anal Biochem ; 178(2): 336-41, 1989 May 01.
Article in English | MEDLINE | ID: mdl-2526606

ABSTRACT

A new procedure for size-dependent fractionation of DNA was investigated. DNA fragments ranging from 10 to 40 kbp were separated by using columns for high-performance gel permeation chromatography. However, the order of elution was opposite to that which would be expected for gel permeation chromatography, i.e., smaller fragments were eluted faster than larger fragments, though separation based on normal gel permeation chromatography was observed when smaller DNA fragments (less than 5 kbp) were applied. The size range of DNA which can be resolved by this new procedure was found to depend on both particle size and flow rate; the use of a column packed with smaller particles or the application of a faster flow rate enabled us to resolve smaller DNA fragments, but the pore size or chemical nature of the column packing had scarcely any effect on the resolution. This mode of separation was attained by using both silica and polymer packings. The results suggest that the separation is based on a hydrodynamic phenomenon.


Subject(s)
Bacteriophage lambda/analysis , DNA, Viral/analysis , Chromatography, Gel , Electrophoresis, Agar Gel
7.
Anal Biochem ; 179(1): 77-85, 1989 May 15.
Article in English | MEDLINE | ID: mdl-2527011

ABSTRACT

An alternative nonradioactive labeling method and a highly sensitive technique for detecting specific DNA sequences are described. The labeling method requires the "Klenow" fragment of DNA polymerase I and random hexanucleotides (synthesized or naturally extracted) as a primer for the production of highly sensitive DNA probes. The system has three main steps: (i) labeling of DNA with biotinylated 11-dUTP; (ii) detection of biotinylated DNA by a one-step procedure with streptavidin-alkaline phosphatase complex; (iii) blocking of background with Tween 20. Twenty attograms (2 X 10(-17) g) of pBR322 plasmid DNA was detected by dot-blot hybridization. Upon Southern blot hybridization, 7.4 fg (7.4 X 10(-15) g) of pBR322 HindIII DNA was detected using the biotinylated pBR322 plasmid DNA probe; 40.8 ag and 7.4 fg of lambda HindIII DNA were detected with the biotinylated whole lambda DNA probe by dot and Southern blot hybridization, respectively. Specific bands were also detected with the biotinylated argininosuccinolyase probe upon Northern blotting of mouse poly(A+) RNA. Further applications for in situ hybridization are also described.


Subject(s)
Biotin/analysis , DNA/analysis , Animals , Bacteriophage lambda/analysis , Blotting, Northern , DNA Probes , DNA, Viral/analysis , Humans , Mice , Nucleic Acid Hybridization , Phosphorus Radioisotopes , RNA, Messenger/analysis
8.
Biochem Pharmacol ; 38(7): 1175-80, 1989 Apr 01.
Article in English | MEDLINE | ID: mdl-2523218

ABSTRACT

1-Methyl-4-phenyl-5-nitrosoimidazole (5NO), which has properties consistent with the biologically active form of a 5-nitroimidazole, was radiolabeled (1-[14C]-methyl) and shown to bind to DNA, but at a rate too slow to account for its bactericidal effect. In the presence of physiological intracellular concentrations of such thiols as glutathione, however, binding was enhanced by 2-3 orders of magnitude, which is quantitatively sufficient to account for the bactericidal effect of 5NO. That 5NO binding was greater for poly[d(G-C).d(G-C)] than for poly[d(A-T).d(A-T)] suggests that the reactive species binds to nucleophilic bases on DNA, a suggestion which is also supported by our finding of a thiol-dependent reaction to form an adduct between 5NO and aniline.


Subject(s)
Carbon Radioisotopes/analysis , DNA, Bacterial/analysis , Imidazoles/metabolism , Nitroimidazoles/analysis , Nitroso Compounds/metabolism , Polynucleotides/analysis , Sulfhydryl Compounds/pharmacology , Bacteriophage lambda/analysis , Cysteine/pharmacology , DNA/analysis , DNA, Bacterial/isolation & purification , Glutathione/pharmacology , Isotope Labeling , Models, Biological , Nitroimidazoles/metabolism , Spectrophotometry, Ultraviolet
10.
Proc Natl Acad Sci U S A ; 85(21): 7882-6, 1988 Nov.
Article in English | MEDLINE | ID: mdl-2973057

ABSTRACT

The activity of the cII protein of phage lambda is probably the critical controlling factor in the choice of the lytic or lysogenic pathway by an infecting virus. Previous work has established that cII activity is regulated through the turnover of cII protein; the products of the hflA and hflB loci of Escherichia coli are needed for a degradative reaction, and lambda cIII functions in stabilizing cII. By using the cloned hflA locus, we have purified a cII-cleaving enzyme that we term HflA. Purified HflA contains three polypeptides; at least two of the subunits are products of the hflA region, and the third is probably a cleavage product of the larger of these two hflA-encoded polypeptides. The HflA protease activity cleaves cII to small fragments. We conclude that the switch between lambda developmental pathways involves regulated cleavage of cII by the specific protease HflA.


Subject(s)
Bacteriophage lambda/analysis , Lysogeny , Serine Endopeptidases/metabolism , Transcription Factors/metabolism , Cloning, Molecular , Escherichia coli/enzymology , Escherichia coli/genetics , Viral Proteins
12.
Anal Biochem ; 175(1): 196-201, 1988 Nov 15.
Article in English | MEDLINE | ID: mdl-2977528

ABSTRACT

A rapid and simple method is described for the isolation of DNA from phage lambda which requires neither special equipment nor expensive material such as cesium chloride for ultracentrifugation nor extractions with organic solvents or ethanol precipitation. Microgram quantities of lambda DNA are obtained in less than 2 h from 90-mm plate lysates or 5-ml liquid cultures. The method allows the simultaneous isolation of large numbers of probes, e.g., clones from phage libraries. Lambda phages are precipitated by polyethylene glycol/sodium chloride and recovered by low speed centrifugation onto glass fiber filters positioned in disposable syringes. The DNA of phages is released by a 50% formamide/4 M sodium perchlorate solution, washed in filter-bound form, eluted with a small volume of low-salt buffer or water, and finally recovered by centrifugation. Comparison of the DNA isolated by this method with that obtained by two conventional procedures reveals both a similar recovery and a similar suitability for restriction enzyme digestion and subcloning.


Subject(s)
Bacteriophage lambda/analysis , DNA, Viral/isolation & purification , Bacteriophage lambda/genetics , Chemical Precipitation , Cloning, Molecular , DNA, Bacterial/isolation & purification , DNA, Viral/genetics , Escherichia coli/analysis , Filtration/instrumentation , Glass
13.
Anal Biochem ; 175(1): 281-3, 1988 Nov 15.
Article in English | MEDLINE | ID: mdl-2977529

ABSTRACT

A procedure for the preparation of DNA from bacteriophage lambda is described, using the Beckman TL-100 bench-top ultracentrifuge. The procedure involves growth of phage in agar plates, precipitation with polyethylene glycol, and a single centrifugation in cesium chloride under conditions that disrupt the phage coat. The method avoids the use of enzymes, ion exchange resins, and phenol. It can be completed in less than a day. The resulting DNA is of good purity and is easily cuttable by restriction enzymes.


Subject(s)
Bacteriophage lambda/analysis , Centrifugation, Density Gradient/methods , Chlorides , DNA, Viral/isolation & purification , Cesium , Chemical Precipitation , Ethanol , Polyethylene Glycols
14.
Biotechniques ; 6(10): 926, 928-9, 1988.
Article in English | MEDLINE | ID: mdl-2856198

ABSTRACT

A procedure is described for the rapid transfer of DNA from agarose gels to nylon membranes using the semi-dry electroblotting technique. A Hind III digest of lambda DNA which was separated in a 1% agarose gel containing Tris, Borate, and EDTA (pH 8.0) was employed for the electrotransfer experiments. Transfer efficiency was determined by staining the DNA on the nylon membranes with a colloidal iron reagent. Current densities of 3-5 mA/sq. cm of gel permitted the transfer of high (23 kb) and low (0.3 kb) molecular weight fragments within 15 min. However, efficient transfer required a high ionic strength buffer that would prevent uneven dehydration of the agarose gel. Critical parameters for the transfer of nucleic acids with the semi-dry technique are discussed.


Subject(s)
DNA, Viral/isolation & purification , Electrophoresis, Agar Gel/methods , Electrophoresis/methods , Bacteriophage lambda/analysis , Biotechnology , Buffers , Endonucleases , Membranes, Artificial , Molecular Weight , Nylons
16.
Science ; 241(4869): 1084-6, 1988 Aug 26.
Article in English | MEDLINE | ID: mdl-2842862

ABSTRACT

Mapping and manipulation of very large genomes, including the human genome, would be facilitated by the availability of a DNA cleavage method with very high site specificity. Therefore, a general method was devised that extends the effective recognition sequences well beyond the present 8-base pair limit by combining the specificity of the restriction endonuclease with that of another sequence-specific protein that binds tightly to DNA. It was shown that the tightly binding lac or lambda repressor protects a restriction site within the operator from specific modification methylases, M.Hha I or M.Hph I, while all other similar sites are methylated and thus rendered uncleavable. A plasmid containing a symmetric lac operator was specifically cleaved by Hha I, only at the site within the operator, after M.Hha I methylation in the presence of the lac repressor, whereas the remaining 31 Hha I sites on this plasmid were methylated and thus not cleaved. Analogous results were obtained with the Hae II site within the lac operator, which was similarly protected by the lac repressor, and with the Hph I site within the phage lambda oL operator, which was protected by lambda repressor from M.Hph I methylation.


Subject(s)
DNA Restriction Enzymes/metabolism , DNA/metabolism , Operator Regions, Genetic , Bacteriophage lambda/analysis , Bacteriophage lambda/genetics , Hot Temperature , Lac Operon , Methylation , Plasmids , Repressor Proteins/metabolism , Substrate Specificity
17.
FEBS Lett ; 235(1-2): 122-4, 1988 Aug 01.
Article in English | MEDLINE | ID: mdl-2969827

ABSTRACT

Single-strand DNA nicks are induced by uranyl nitrate or uranyl acetate in combination with long-wavelength (lambda approximately 420 nm) ultraviolet irradiation. The nicks occur randomly with respect to the DNA sequence. Using the lambda-repressor/ORI operator DNA system it is shown that uranyl salts can be used to photofootprint protein contacts with the DNA backbone.


Subject(s)
DNA/metabolism , Organometallic Compounds/pharmacology , Proteins/metabolism , Ultraviolet Rays , Uranium/pharmacology , Uranyl Nitrate/pharmacology , Bacteriophage lambda/analysis , Base Sequence , DNA/drug effects , DNA/radiation effects , DNA, Superhelical/drug effects , DNA, Superhelical/metabolism , DNA, Superhelical/radiation effects , Electrophoresis, Polyacrylamide Gel , Operator Regions, Genetic , Photochemistry , Plasmids , Repressor Proteins/metabolism
18.
FEBS Lett ; 234(1): 56-60, 1988 Jul 04.
Article in English | MEDLINE | ID: mdl-2968919

ABSTRACT

The ability of the isolated carboxy-terminal domain of the LexA repressor of Escherichia coli to form dimers and tetramers has been investigated by equilibrium ultracentrifugation. This domain, that comprises the amino acids 85-202, is readily purified after self-cleavage of the LexA repressor at alkaline pH. It turns out that the carboxy-terminal domain forms dimers and tetramers essentially as the entire LexA repressor. The corresponding association constants were determined after non-linear least squares fitting of the experimental concentration distribution. A dimer association constant of K2 = 3 X 10(4) M-1 and a tetramer association constant of K4 = 2 X 10(4) M-1 have been determined. Similar measurements on the entire LexA repressor [(1985) Biochemistry 24, 2812-2818] gave values of K2 = 2.1 X 10(4) M-1 and K4 = 7.7 X 10(4) M-1. Within experimental error the dimer formation constant of the carboxy-terminal domain may be considered to be the same as that of the entire repressor whereas the isolated domain forms tetramers slightly less efficiently. It should be stressed that the potential error in K4 is higher than that in K2. The overall conclusion is that the two structural domains of LexA have also well-defined functional roles: the amino-terminal domain interacts with DNA and the carboxy-terminal domain is involved in dimerisation reinforcing in this way the binding of the LexA repressor to operator DNA.


Subject(s)
Bacterial Proteins , Escherichia coli/analysis , Serine Endopeptidases , Bacteriophage lambda/analysis , Macromolecular Substances , Repressor Proteins , Ultracentrifugation
20.
Anal Biochem ; 168(2): 324-31, 1988 Feb 01.
Article in English | MEDLINE | ID: mdl-2834980

ABSTRACT

A simple and rapid procedure for purifying large quantities of bacteriophage lambda particles and DNA is described. The procedure involves DEAE-cellulose column chromatography of the phage particles and elution of the phage particles from the column with a low-ionic-strength buffer. The resulting phage were well separated from RNA, DNA, and proteins derived from Escherichia coli host cells. The lambda DNA was prepared from the purified phage particles by the conventional method of phenol extraction and ethanol precipitation. This procedure did not use nucleases, proteases, detergents, or CsCl density gradient centrifugation. The lambda DNA obtained by this method was equivalent in purity to the material prepared by CsCl density gradient centrifugation and amenable to restriction enzyme digestion, ligation, radiolabeling, and double-stranded DNA sequencing. A detailed protocol is described for obtaining 0.5 to 1.0 mg DNA from a 1-liter liquid lysate in less than 5 h. This procedure is simple, inexpensive, and timesaving, and is particularly suitable for large-scale isolation of lambda DNA.


Subject(s)
Bacteriophage lambda/analysis , DNA, Viral/isolation & purification , Chromatography, DEAE-Cellulose , DNA Restriction Enzymes/pharmacology , Electrophoresis
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