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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.

We have purified the phage lambda transcriptional activator protein cII. The procedure described allows cII to be obtained in both high purity and yield, and thus allows detailed physical and chemical analysis. We demonstrate that cII in solution is a tetrameric protein and that it undergoes specific processing at its NH2-terminal end. In addition, the protein is characterized as to its molar extinction coefficient, molecular weight, amino acid composition, isoelectric point, alpha-helical content, and antigenic capability.

A restriction endonuclease Eco72I with a novel substrate specificity has been isolated from Escherichia coli strain RFL 72. The enzyme recognizes (Formula: see text) hexanucleotide palindromic sequence and cleaves it, as indicated by the arrows, to produce blunt-ended fragments.

We have investigated internal motion of DNA in bacteriophages by measuring fluorescence anisotropy decays of intercalated ethidium. The results showed large suppression of the internal motion of the inner DNA; the interhelix interaction of the DNA in the phage head is considered to enhance the effective viscosity of the DNA rod and to restrict the angle of the internal motion. Considering that the observed internal motion arises mainly from torsional motion of the DNA, we have calculated the movable angles of the torsional motion (the standard deviation of the torsional motion) of the DNA in the phage heads. The magnitude of the calculated movable angles indicates the extent of suppression of the DNA movement in the phage head; in lambda wild type phage, the DNA is packed most rigidly in the head and the motion is found to be restricted most severely. In a deletion mutant of lambda phage, whose inner DNA content is deficient by 17.6%, steric hindrance from the interhelix DNA interaction is decreased, and the DNA can move more easily. In T4 wild type phage, although the extent of condensation of the inner DNA is the same as that in lambda wild type phage, the DNA was fairly mobile. The presence of glucosylated hydroxymethylcytosine is suggested to influence the rigidity of the inner DNA or packaging mode of the DNA in the T4 head.

The morphogenesis of bacteriophage lambda proheads is under the control of the four phage genes B, C, Nu3 and E, and the two Escherichia coli genes groEL and groES . It has been shown previously that extracts prepared from cells infected with a lambda C-E- mutant accumulate a gpB polymer, which behaves as a biologically active intermediate in prohead assembly. This gpB activity has been called a preconnector , as it is probably a precursor to the head-tail connector. We now report the partial purification of biologically active preconnectors and the characterization of its structure. In the electron microscope, preconnectors appear as donut -like structures composed of several subunits displaying radial symmetry. Optical filtration of periodic arrays of preconnectors showed that the structure has 12-fold rotational symmetry. Side views of the preconnector reveal that it resembles an asymmetrical dumbell . This information has been used to construct a three-dimensional model of the preconnector . The implications of this structure for prohead shape and function, and for DNA packaging are discussed.

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.

We have developed a new DNA-DNA crosslinking strategy based on a cleavable bispsoralen reagent and used this strategy to study the structures of bacteriophage lambda and the animal virus SV40. Our results show that in both lambda and SV40, all restriction fragments examined can be crosslinked to all other restriction fragments. In bacteriophage lambda, the crosslinking data are consistent with a random packaging model, while in SV40 there is some deviation from the random model. These results imply that the structures of DNA inside these viruses are either highly disordered or very complex.

The dependence of irreversible structural changes in phage lambda DNA on the degree of its protonation in a solution and inside the virion has been found by measuring the transfection activity of bacteriophage. The different effect of ionic strength on pH-dependence of the irreversible changes in the structure of DNA upon its protonation in a solution or in situ has been registered and explained. The insignificant shift of pH from neutral region value in 0.1 M NaCl has resulted in a damaging effect of H+ ions on compact DNA in situ as compared to the DNA in a solution. The effect of H+ ions on compact DNA in situ is mainly based on the formation of noncovalent intermolecular DNA-protein and DNA-DNA linkages.

The dnaA gene of Escherichia coli K-12, supposedly present in the deoxyribonucleic acid (DNA) of specialized transducing phase lambda i21 dnaA-2, was cloned onto plasmid pBR322. The new plasmid was named pMCR501. Physical analyses of DNAs of lambda i21 dnaA-2 and pMCR501 revealed the following. The lambda i21 dnaA-2 DNA retained the delta sr I lambda 1-2 and ninR5 deletions and imm21 substitution which were originally present in the parental phage. The size reduction was compensated for by the insertion-substitution segment (tna-dnaA region) in lambda i21 dnaA-2 DNA. The fractional size of this segment was approximately 7 megadaltons (Md), or 10 kilobases, which was found to be the sum of the tna insertion subsegment of ca. 3.5 Md and the dnaA substitution subsegment of ca. 3.5 Md. Phage P1-mediated transductional mapping between the dnaA46 and tna mutations gave a cotransduction frequency of 84%, corresponding to approximately 5 kilobases. Thus, it is strongly suggested that the dnaA gene resides in the lambda i21 dnaA-2 DNA. Cleavage mapping with the restriction endonuclease of pMCR501 DNA confirmed that it was constructed by excising a BamHI fragment of 4.29 Md, containing the 3.5-Md dnaA substitution segment, from the lambda i21 dnaA-2 DNA, inserting it into the sole BamHI cleavage site on pBR322.

DNA of lambda and seven related phages was digested with restriction endonuclease, EcoRI. Seven different fragment patterns were observed, only two of the eight phages showing identical profiles. Restriction enzyme fragment analysis is thus shown to be a sensitive tool for the differentiation of biologically indistinguishable phages.

A method that provides an easy, rapid, and reproducible way for separating closed circular DNA species from linear DNA and nicked circles is described. The method is based on the difference in mobility of form I (supercoiled) DNA and form II (nicked circles), and the differential mobility of relaxed circular DNA in the presence and absence of ethidium bromide (EtdBr). It can be used for detection or for purification of plasmid, episomal, or viral DNA from the bulk of cellular DNA, or from other DNA mixtures.

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.

The DNA sequence of the int and xis genes of lambda bacteriophage, as well as that of the PI promoter and a region of unknown function beyond this, was determined by the chain termination procedure. The Pribnow box sequence of the PI promoter lies just within the xis gene, and both possible sites of mRNA initiation from PI are within the xis gene. The end of the xis gene in its turn overlaps the start of the int gene by 23 base pairs, in a different reading frame. This overlap may play a role in ensuring efficient excision of the prophage in response to natural induction signals.

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.

NOESY spectroscopy at 500 HMz was employed to assign resonances of nonexchangeable protons in the 1H NMR spectrum of the synthesized 17 base pair duplex of deoxyribonucleotides comprising the OR1 operator, one of the specific binding sites for the bacteriophage lambda cro repressor. A pure absorption spectrum that was obtained by the phase sensitive detection technique allowed to perform a resonance assignment of base and deoxyribose protons, excepting H-5'- and H-5"-protons, on the basis of a single NOESY experiment, mixing time 200 ms. The sequential assignment strategy for 2D NMR spectra of oligonucleotides was used for this purpose. The data obtained and the previous results on OR3 are discussed in the view of the conformation of operators in solution. It is shown that both duplexes exist in the DNA B-form with its intrinsic anti conformation of the nucleotides. Conformation of DNA segments with identical primary structures are similar, and local deviations from the classical B-form are determined by a nucleotide sequence.

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.

Excellent resolution of chromosomal DNA molecules from Saccharomyces cerevisiae, Candida albicans and Schizosaccharomyces pombe has been obtained using alternating contour-clamped homogeneous electric field (CHEF) gel electrophoresis. The largest of these molecules is greater than 5 Mb in size and is resolved after 130 hours in a 0.6% agarose gel at a field strength of 1.3 V/cm and a switching interval of 1 hour. Separation of concatamers of phage lambda DNA reveals four regions of resolution in alternating CHEF gel electrophoresis. There are two regions of good resolution in which mobility approximates a linear function of molecular weight. These are separated by a region of lower resolution and bounded at high molecular weights by a region of little or no resolution. The four regions are of practical and possibly theoretical importance.

Bromoacetaldehyde (BAA) was used to study the secondary structure of DNA in lambda-phage particles. It was determined that about 1% of the adenines in the intraphage lambda-DNA reacts readily with BAA, thus, they are placed in DNA sites with disturbed complementary interactions. These adenines are close to the tryptophan residues of the phage protein. Fluorescence emission of epsilon A in the intraphage DNA is dramatically quenched. This, apparently, indicates the interaction between epsilon A and Trp- and/or Tyr- and/or Met-residues of phage protein.

The structure of a bacteriophage lambda cro repressor hydrophobic globule was studied by the technique of 1H NMR spectroscopy at 500 MHz. The analysis of NOE difference spectra and building of the molecular models for the most probable fragments of the secondary structure allowed us to assign many signals in the protein spectrum and to identify the intramolecular interactions which stabilized the hydrophobic globule and the tertiary structure of the molecule. The results suggest that the structure of the cro repressor hydrophobic globule in solution coincides with the crystal one, although there are some differences in the mutual arrangement of the alpha 1 helix and the three-fold beta-sheet. Resonance assignment has made it possible to study conformational changes and specific interactions of the cro repressor by using suitable reporter groups.

Under anaerobic conditions where the nitroaromatic radiation-sensitizer misonidazole substitutes for dioxygen, DNA strand breakage (gaps with phosphate residues at each end) by the nonprotein chromophore of the antitumor antibiotic neocarzinostatin (NCS-Chrom) is associated with the generation of a reactive form of formate from the C-5' of deoxyribose of thymidylate residues. Such lesions account for a minority (10-15%) of the strand breakage found in the aerobic reaction without misonidazole. Amino-containing nucleophiles such as tris(hydroxymethyl)aminomethane (Tris) and hydroxylamine act as acceptors for the activated formate. The amount of [3H]formyl hydroxamate produced from DNA labeled with [5'-3H]thymidine is comparable to the spontaneously released thymine. During the course of the reaction, misonidazole undergoes a DNA-dependent reduction and subsequent conjugation with glutathione used to activate NCS-Chrom. From these and earlier results, we propose a possible mechanism in which the carbon-centered radical formed at C-5' by hydrogen atom abstraction by thiol-activated NCS-Chrom reacts anaerobically with misonidazole to form a nitroxyl-radical-adduct intermediate, which fragments to produce an oxy radical at C-5'. beta-Fragmentation results in cleavage between C-5' and C-4' with the generation of 3'-formyl phosphate-ended DNA, a high-energy form of formate, which spontaneously hydrolyzes, releasing formate and creating a 3'-phosphate end, or transfers the formyl moiety to available nucleophiles. A similar mechanism, involving dioxygen addition, is probably responsible for the 10-15% DNA gap formation in the aerobic reaction.

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.

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.

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.

A new method for preparing small quantities of lambda DNA from phage lysates has been developed. The protocol is based on the concentration and purification of bacteriophage particles from crude lysates using small DEAE-cellulose columns. This chromatographic step gives an absolute separation of the lambda DNA from the cellular nucleic acids and a 20-fold enrichment relative to the major soluble proteins in crude lysates, while effecting a 10-fold concentration of the phage. Final deproteinization and concentration of the lambda DNA is achieved by conventional precipitation steps. The lambda DNA produced by this method is shown to be nondegraded, biologically active, and an excellent substrate for restriction enzymes. A detailed protocol is provided for starting with individual plaques and using the method to obtain purified DNA from large numbers of lambda clones.

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.

A simple and rapid (1 day) method for preparation of lambda phage DNA was proposed. The method included two main steps: (a) growth and lysis of bacteria containing lambda phage and (b) purification of lambda phage DNA by hydroxylapatite chromatography. The phage DNA prepared by this method was intact and free of RNA, proteins, and bacterial DNA.

The mapping of the sites of cleavage of nine restriction endonucleases (EcoRI, HindIII, BamHI, SalI, KpnI, SstI, BglII, XhoI, and XbaI) on 21 Charon phage vectors is described. Maps of individual subsections were obtained and then combined to assemble the complete vector maps. Calculations of maximum and minimum sizes of inserts which may be carried by the vectors using different restriction endonucleases or pairs of restriction endonucleases are presented. The regions mapped include several parts of phi 80 that had not been mapped previously.

Phage lambda DNA was covalently coupled to epoxy-activated cellulose to form a stable DNA-cellulose matrix for affinity chromatography of sequence-specific DNA-binding proteins. The accessibility of three specific six-base sequences, GGATCC (BamHI), GAATTC (EcoRI) and AAGCTT (HindIII) was studied quantitatively and qualitatively by restriction analysis followed by labelling of their recessed ends. All sites are randomly accessible. The site accessibility is variable, BamHI greater than HindIII greater than EcoRI, and within the range 20-100% depending on base composition and internal structure of the sequence. DNA-epoxycellulose, because of its high efficiency of coupling, capacity, stability and accessibility, can be of great help in the isolation and characterization of sequence-specific DNA-binding proteins.

Data are described on identification, isolation and purification of restricting endonuclease Sau 6782 as well as on estimation of the enzyme recognition site. Conditions were developed for growing of Staphylococcus aureus 6782 strain, which enabled to produce a maximal yield of the restricting activity containing minimal level of nucleases. The procedure for isolation and purification of restrictase Sau 6782 involved affinity chromatography on Blue Sepharose and cation exchange chromatography on phosphocellulose PII. The enzyme preparation obtained was free from impurities of unspecific nucleases. The yield of the Sau 6782 restrictase constituted 1,000 un from 1 g of the culture cells. Restrictase Sau 6782 recognized the nucleotide sequence 5'...GATC...3' and was the isoshizomere of the Sau 3A enzyme.

The N protein (pN) specified by bacteriophage lambda is an antitermination factor and is required for phage development. pN can be assayed by making use of the observation that the in vitro synthesis of trp mRNA in a reaction programmed with DNA template from lambda trp transducing phage bearing N- and fed- mutations is pN dependent (Ishii et al., 1980). The assay has been used to purify pN. We have observed that pN forms a complex with E. coli protein(s) and is dissociated in the presence of urea. The complex is not formed in host bacteria bearing the nusA-nusB- mutations. pN is a basic protein and heat-stable. Using these characteristics, we have purified pN to virtual homogeneity as judged by polyacrylamide gel electrophoresis in the presence of SDS. pN is a monomeric protein and its mol. wt. is approx. 14 000. The antiterminating activity of pN appears to be enhanced by complex formation with host-encoded protein(s) depending on the nusA and/or nusB gene function.

A chromogenic method using biotinylated secondary antibodies and peroxidase-coupled avidin for screening cDNA expression libraries is described. This method offers increased sensitivity over peroxidase-coupled secondary antibodies and rapid processing of samples, and avoids preparation and handling of radioactive materials. All materials for the chromogenic assay are available commercially and the method offers a fast and easy way to screen lambda and plasmid expression libraries with mono- and polyclonal antibodies.

Experiments are reported which bear on two spool models proposed for packaging the DNA of phage lambda. Both spool models fill an assumed spherical cavity with DNA wrapped in cylindrical or quasi-cylindrical layers composed of adjacent circular turns. In the curved-spool model, a single continuous segment of DNA, about 20% of the DNA length and probably located near the left end of the DNA, is in contact with the coat protein of the phage capsid. In the straight spool model, there are several DNA segments in contact with the capsid; they are concentrated in one half (probably the left half) of lambda DNA. We have identified the loci on the DNA which are in contact with the capsid by chemical crosslinking, induced by ultraviolet-irradiation of phage containing 5-bromodeoxyuridine in place of thymine. In an electron microscope experiment, phage are first lysed with EDTA, and then spread in a cytochrome c film by the formamide method. The disrupted capsid, which has the appearance of a phage ghost, serves as a marker showing where the DNA is crosslinked to the coat. The left end of the DNA is not distinguished from the right end, and so the map of DNA-capsid contacts is folded over on itself. Contacts are found nearly randomly over the entire map. In a second experiment, DNA from lysed, crosslinked phage is cut either with EcoRI or HindIII restriction endonucleases and the cut restriction fragments are labeled at their ends with 32P. Density centrifugation in a CsCl gradient separates free DNA from restriction fragments crosslinked to protein. After digestion with proteinase k, the DNA fragments previously crosslinked to protein are identified by size after agarose gel electrophoresis. DNA fragments from all parts of the genome are found. These two experiments show that, if the DNA of each phage is packaged identically, then the curved-spool model is ruled out and the straight spool model is unlikely. Alternatively, the manner of packaging the DNA may vary from one phage to the next. These results agree with other recent experiments on lambda DNA packaging by Hall & Schellman (1982a,b), and by Haas et al. (1982). A different experiment is also reported. The psoralen derivative aminomethyltrioxalen (AMT) is allowed to intercalate into lambda phage and then the DNA strands are crosslinked by ultraviolet-irradiation after the rapid phase of AMT intercalation is complete. The DNA is subsequently denatured by glyoxal modification and spread for electron microscopy in a cytochrome c film by the formamide method.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Relationships between DNA length and electrophoretic mobility in an agarose gel have been compared by estimating the lengths of known DNA polymer fragments, using other fragments in the series as standards. Global estimates were made using 10 fragments as standards; local estimates were made using the two closest fragments on either side of the unknown as standards. Most relationships were fitted by least squares. All the relationships gave more accurate local than global estimates. The most accurate results were obtained using the reciprocal relationship, where the maximum error in the local estimates was less than 0.1%. The semilog relationship gave the least accurate results, with a maximum error in the local estimates of almost 5%. The polymer fragments were also used as standards to estimate the lengths of lambda DNA restriction fragments. Here the estimates were in error by up to 3%, indicating the influence of base composition and sequence on electrophoretic mobility.

This article describes the construction of an inexpensive and reliable data acquisition system for a Varian E-line Century Series ESR spectrometer utilizing an Apple II Plus microcomputer. All necessary hardware is readily available and used without modification. A BASIC program for routine collection, display, plotting and disk storage of experimental data has been written and subsequently compiled into machine code for high speed operation. The interface offers distinct advantages in spectral resolution as well as instrument control. An example of signal enhancement via computer controlled time averaging is presented for a spin labeled DNA experiment. The technique has recently been applied to studies of relative binding affinities of gene-32 protein for various spin-labeled polynucleotides.

The three-dimensional structures of cro repressor protein and of the amino-terminal domain of lambda repressor protein, both from bacteriophage lambda, are compared. The second and third alpha-helices, alpha 2 and alpha 3, are shown to have essentially identical conformations in the two proteins, confirming the significance of the amino acid sequence homology previously noted between these and other DNA binding proteins in the region corresponding to these helices. The correspondence between the two-helical units in cro and lambda repressor protein is better than the striking agreement noted previously between two-helical units in cro and catabolite gene-activator protein. Parts of the first alpha-helices of repressor and cro show a structural correspondence that suggests a revised sequence homology between the two proteins in their extreme amino-terminal regions. In particular, there is a short loop between the alpha 1 and alpha 2 helices of lambda repressor that is missing from cro. This structural difference may account for the observed differences found with different cros and repressors in the pattern of phosphates whose ethylation prevents the binding of these proteins to their specific recognition sites. Although the two proteins have strikingly similar alpha 2-alpha 3 helical units that are presumed to bind to DNA in an essentially similar manner, stereochemical restrictions prevent the alpha 2-alpha 3 units of the respective proteins aligning on the DNA in exactly the same way.

Supercoiled Harvey sarcoma virus (Ha-SV) DNA was extracted from newly infected cells by the Hirt procedure, enriched by preparative agarose gel electrophoresis, and digested with EcoRI, which cleaved the viral DNA at a unique site. The linearized Ha-SV DNA was then inserted into lambda gtWESlambda B at the EcoRI site and cloned in an approved EK2 host. Ha-SV DNA inserts from six independently derived recombinant clones have been analyzed by restriction endonuclease digestion, molecular hybridization, electron microscopy, and infectivity. Four of the Ha-SV DNA inserts were identical, contained about 6.0 kilobase pairs (kbp), and comigrated in agarose gels with the infectious, unintegrated, linear Ha-SV DNA. One insert was approximately 0.65 kbp smaller (5.35 kbp) and one was approximately 0.65 kpb larger (6.65 kpb) than the 6.0 kpb inserts. R-looping with Ha-SV RNA revealed that the small (5.35 kbp) insert contained one copy of the Ha-SV RNA. Preliminary restriction endonuclease digestion of the recombinant DNAs suggested that the middle-size inserts contained a 0.65-kbp tandem duplication of sequences present only one in the small-size insert; this duplication corresponded to the 0.65-kpb terminal duplication of the unintegrated linear Ha-SV DNA. The large-size insert apparently contained a tandem triplication of these terminally located sequences. DNA of all three sized inserts induced foci in NIH 3T3 cells, and focus-forming activity could be rescued from the transformed cells by superinfection with helper virus. Infectivity followed single-hit kinetics, suggesting that the foci were induced by a single molecule.