Lambda DNA Fragments Research Articles

Relaxation effects in the gel electrophoresis of DNA in intermittent fields

The electrophoretic mobility of restriction fragments of lambda DNA in agarose gels declines if the field is intermittent rather than continuous, with a greater effect on the longer fragments. The changes are compatible with the assumption of two exponential relaxation processes for field-dependent configurational changes, one when the field is turned on and another when it terminates. The length dependence at the extrapolated limit of mobility for short pulses with long intervals corresponds closely to the simple inverse proportionality to length expected from theoretical considerations when the molecular configuration is not affected by the electric field. Simple intermittent fields would allow separation of longer molecules than can ordinarily be resolved. The relaxation times for both the change in conformation imposed by the field and the return to field-free conformation vary as approximately the second power of the length of the molecule, independent of the salt concentration or field strength and varying only slightly with gel density. These relations are not in good agreement with properties expected from reputation theory, and they suggest that a different mechanism must be invoked for the electrophoretic migration of long DNA molecules at ordinary values of field strength.

Occurrence and partial characterization of an RNA species in rice mitochondria

An unusual species of RNA, designated RL-1, was detected in rice ( Oryza sativa subsp. Japonica Li-ming A, CMS-Chinsurah Boro II type) mitochondria. Its molecular length was estimated to be 18 kb by using Bam HI fragments of Lambda DNA as markers. RL-1 RNA was not significantly affected by DNase I or by nuclease S1. It was not digested by RNase A under high salt conditions, but was completely digested under low salt conditions. When the total mitochondrial nucleic acids in 15% ethanol-saline-tris-EDTA (STE) solution were loaded on a microcrystalline cellulose chromatographic column, the mtDNA and low molecular RNAs were eluted by 15% ethanol-STE solution, but RL-1 molecules remained bound to the cellulose and were then eluted with STE buffer without ethanol. These results show that the RL-1 molecule may be double-stranded. Among the other ten rice cultivars surveyed, 18 kb RNA was detected in four all belonging to the same CMS-Chinsurah Boro II type (like Li-ming A), but not in five CMS-lines of rice of non-Chinsurah Boro II type or in fertile cultivar IR-24.

The interaction ofE.coliintegration host factor and λcosDNA: multiple complex formation and protein-induced bending

The interaction of E. coli's integration Host Factor (IHF) with fragments of lambda DNA containing the cos site has been studied by gel-mobility retardation and electron microscopy. The cos fragment used in the mobility assays is 398 bp and spans a region from 48,298 to 194 on the lambda chromosome. Several different complexes of IHF with this fragment can be distinguished by their differential mobility on polyacrylamide gels. Relative band intensities indicate that the formation of a complex between IHF and this DNA fragment has an equilibrium binding constant of the same magnitude as DNA fragments containing lambda's attP site. Gel-mobility retardation and electron microscopy have been employed to show that IHF sharply bends DNA near cos and to map the bending site. The protein-induced bend is near an intrinsic bend due to DNA sequence. The position of the bend suggests that IHF's role in lambda DNA packaging may be the enhancement of terminase binding/cos cutting by manipulating DNA structure.

Evaluation of three procedures using a laser densitometer and microcomputer for estimating molecular sizes of restriction endonuclease digest fragments and application to Campylobacter jejuni chromosomal DNA

Laser densitometer scanning and three methods of computation were investigated for estimating sizes of restriction endonuclease digest fragments of Campylobacter jejuni chromosomal DNA. Accuracy of the procedures was determined with standard bacteriophage lambda DNA fragments. The program DNASIZE was the most accurate for fragments between 2.3 and 33.5 kb while the program DNAFRAG and multiple regression analysis gave slightly greater errors particularly for smaller fragments. Hae III fingerprints of DNA from six C. jejuni strains contained at least 25 resolvable fragments with sizes between 3.2 and 35.8 kb estimated by DNASIZE and several differences between pattern types were detected.

Role of homology and pathway specificity for recombination between plasmids and bacteriophage lambda.

To determine the minimum amount of homology required for efficient recombination in Escherichia coli, we measured recombination frequencies between bacteriophage lambda and pBR322 derivatives containing lambda DNA fragments of various sizes by assaying for phages that could transduce the bla and ori genes of pBR322. Efficient recombination required about 40 bp of homology; increases in homology above 40 bp resulted in proportionate increases in recombination, while decreases below 40 bp resulted in precipitous decreases in recombination. The recA+ gene stimulated recombination over the entire range of homologies tested. Restriction enzyme digests of several recombinant DNA molecules indicated that they contained the complete plasmid DNA inserted in the lambda genome as expected for a reciprocal crossover. Analysis of recombination frequencies in different recombination-deficient mutant strains indicated that the formation of lambda-plasmid cointegrates by homologous recombination proceeded predominantly by the RecBC pathway and very inefficiently, if at all, by the RecE and RecF pathways.

Determination of DNA sequences containing methylcytosine in Bacillus subtilis Marburg.

The methylcytosine-containing sequences in the DNA of Bacillus subtilis 168 Marburg (restriction-modification type BsuM) were determined by three different methods: (i) examination of in vivo-methylated DNA by restriction enzyme digestion and, whenever possible, analysis for methylcytosine at the 5' end; (ii) methylation in vitro of unmethylated DNA with B. subtilis DNA methyltransferase and determination of the methylated sites; and (iii) the methylatability of unmethylated DNA by B. subtilis methyltransferase after potential sites have been destroyed by digestion with restriction endonucleases. The results obtained by these methods, taken together, show that methylcytosine was present only within the sequence 5'-TCGA-3'. The presence of methylcytosine at the 5' end of the DNA fragments generated by restriction endonuclease AsuII digestion and the fact that in vivo-methylated DNA could not be digested by the enzyme XhoI showed that the recognition sequences of these two enzymes contained methylcytosine. As these two enzymes recognized a similar sequence containing a 5' pyrimidine (Py) and a 3' purine (Pu), 5'-PyTCGAPu-3', the possibility that methylcytosine is present in the complementary sequences 5'-TTCGAG-3' and 5'-CTCGAA-3' was postulated. This was verified by the methylation in vitro, with B. subtilis enzyme, of a 2.6-kilobase fragment of lambda DNA containing two such sites and devoid of AsuII or XhoI recognition sequences. By analyzing the methylatable sites, it was found that in one of the two PyTCGAPu sequences, cytosine was methylated in vitro in both DNA strands. It is concluded that the sequence 5'-PyTCGAPu-3' is methylated by the DNA methyltransferase (of cytosine) of B. subtilis Marburg.

Thermosensitivity of a DNA recognition site: activity of a truncated nutL antiterminator of coliphage lambda.

Antitermination is an important transcriptional control. In bacteriophage lambda, the presence of the nut antiterminators between the promoters and terminators results in relatively unhindered transcription when the lambda N gene product and necessary host factors are supplied. This antitermination system has been rendered thermosensitivity by modification of the nut site. A fragment of lambda DNA [74 base pairs (bp) in length]that contained the 17-bp nutL core sequence, but lacked the 8-bp boxA sequence, was cloned in a pp-N-tL1-galK plasmid between the pp promoter and gene N. This fragment mediated antitermination of transcription at 30 degrees C, as measured by assaying galK gene expression in Escherichia coli. At 42 degrees C, however, antitermination at the lambda tL1 terminator was abolished. Antitermination at 42 degrees C was restored by replacing the 74-bp nutL fragment with longer sequences containing both nutL and boxA or by cloning a synthetic boxA sequence ahead of the 74-bp nutL fragment. Thus, efficient antitermination required both boxA and the 17-bp nutL core, with the latter becoming conditionally defective when the boxA sequence was deleted.

Origin and initiation sites of lambda dv DNA replication in vitro.

The sequence of lambda DNA essential for the unique initiation of replication was analyzed in an in vitro replication system. Fragments of lambda DNA were inserted into pBR322 and used as templates or were circularized in vitro in the absence of pBR322 and employed in the same tests. A 165-bp region left of the EcoRI site in the O gene of the lambda genome was defined as the functional origin. This region, which we defined as the ori region, carries, in order, the 4 19-bp repeat sequences where the O protein binds (ori-repeats), an A+T-rich stretch, and a region that constitutes part of a large palindromic structure. Regions that have long been suspected to participate in lambda DNA replication initiation, ice and oop were not required for the O, P-dependent lambda-specific replication initiation. The lambda dv and the "ori region plus" recombinant plasmids initiated DNA synthesis at or around this region, and the reaction depended on the presence of lambda-coded initiators, O and P proteins. Early replicative intermediates of lambda dv were prepared in an in vitro replication system in the presence of ddCTP, an inhibitor of DNA chain elongation. This system allows only that DNA synthesis that is a result of replication initiation. Using this system, the initiation site(s) of the DNA synthesis was finely analyzed by mapping the transition sites from primer RNA to DNA synthesis. Short-chain DNAs produced from regions near the ori region were purified from the intermediates. A fraction of the short-chain DNAs was covalently linked to primer RNA. The 5'-ends that had been linked to RNA (transition sites) were exposed by alkaline hydrolysis, labeled with 32P, and the transition sites were mapped along the nucleotide sequence of the genome. Two striking features emerged from this analysis: (i) The transition sites are located on both sides of the ori region, and no transition arose within the 165-bp ori region; (ii) The transition sites on both sides are not unique, but multiple, and are clustered in one of the 2 strands. Furthermore, their orientation demonstrates that the DNA synthesis in initiation of replication from the 2 sides of the ori region converge. The frequency of the "leftward" DNA synthesis is several times higher than that of "rightward" synthesis. These results reflect asymmetric bidirectional replication of lambda dv DNA, and may also reflect replication of lambda phage DNA.

Macromolecular crowding accelerates the cohesion of DNA fragments with complementary termini.

Macromolecular crowding increases the rate of nonenzymatic cohesion of the complementary ends of lambda DNA. Both lambda DNA and DNA fragments bearing the cohesive ends of lambda DNA are similarly affected. High concentrations of plasma albumin or Ficoll 70 increase the rate of cohesion by ca. 100-fold whereas high concentrations of polyethylene glycol 8000 cause greater than 2000-fold stimulation in this rate. These results have implications for the mechanism of polymer-stimulated enzymatic ligation of DNA or RNA. In addition, these crowding effects may help to explain the rapid cohesion of lambda DNA observed in vivo. An improved procedure for the recovery of DNA fragments separated by agarose gel electrophoresis is also described.

DNA sequences necessary for packaging of bacteriophage lambda DNA.

The extent of DNA flanking the "cohered cohesive end" site of bacteriophage lambda DNA, which is required for packaging, was determined by using defined DNA fragments and a cosmid in vivo packaging assay. From the right end of lambda DNA a 20- to 36-base-pair stretch extending from the center of the cohered cohesive ends is shown to be required, whereas the packaging efficiency of cosmids extending to 70 base pairs into the left lambda arm is reduced to 10% (compared to a fragment extending until about 80 base pairs). A 60-base-pair stretch of the left arm leaves an efficiency of only 1%. The segment thus delineated, by the nature of the assay, is both necessary and sufficient for the binding of packaging proteins to the DNA, the packaging of DNA itself, the DNA cleavage, and successful injection of the DNA into a bacterial host. By contrast, in vitro packaging of restriction fragments of mature lambda DNA directly demonstrated the selectivity of the packaging proteins for the fragment originating from the left end of the DNA. The results of the two complementary experiments are discussed in terms of the various steps before, during, and after packaging for which different sequences flanking and including the cohered cohesive ends might be required.

Construction and characterization of plasmid vectors for cloning in Staphylococcus aureus and Staphylococcus carnosus.

Several plasmid vectors for cloning in Staphylococcus aureus and S. carnosus have been constructed and characterized. The chimeric plasmids are composed of parts of the following parental plasmids: The chloramphenicol-resistance plasmid, pC194, the tetracycline-resistance plasmid, pMK148, and the erythromycin-resistance plasmid, pE12. All the chimeric plasmids confer two selectable antibiotic-resistance markers on host cells. Insertional inactivation of the various antibiotic-resistance markers occurred at the BclI site of pE12, and the Sau96- or AvaII-site of pMK148; only a slight inactivation of the chloramphenicol-resistance marker occurred at the HaeIII-site of pC194. The chimeric plasmids pCT20 and pCE10 are both stable in S. aureus and S. carnosus. In addition, the hybrid plasmids of pCT20 and pCE10, containing lambda-DNA fragments in various restriction sites between 0.4 and 1.2 kb, are stably maintained. The inserted lambda-DNA fragments appear unchanged.

Comparison of three methods for measuring locations of long range base pairing in single-stranded DNA by electron microscopy

A comparison was made of three procedures used for measuring the locations of long range base pairing in single-stranded DNA. All three procedures gave equivalent results for the location of a transposon, Tn903, and for a loop created by pairing between the nut and operator segments on an Eco RI restriction fragment of lambda DNA. DNA molecules prepared by a T4 gene 32 protein procedure had the best contrast. The highest frequency of measurable loops resulted from a procedure which utilized ammonium acetate/formamide spreading conditions. This procedure also appeared to be the simplest and most satisfactory procedure. Only one procedure revealed the fact that the nut r and o r regions are separated by several hundred bases, but this procedure, which involves crosslinking with trioxsalen, gave generally unsatisfactory DNA morphology. None of the procedures revealed the existence of a short “bulge” loop of single-stranded DNA located between nut l and o l and a longer “bulge” loop located between nut r and o r .

Detection and mapping of homologous, repeated and amplified DNA sequences by DNA renaturation in agarose gels.

A new molecular hybridization approach to the analysis of complex genomes has been developed. Tracer and driver DNAs were digested with the same restriction enzyme(s), and tracer DNA was labeled with 32P using T4 DNA polymerase. Tracer DNA was mixed with an excess amount of driver, and the mixture was electrophoresed in an agarose gel. Following electrophoresis, DNA was alkali-denatured in situ and allowed to reanneal in the gel, so that tracer DNA fragments could hybridize to the driver only when homologous driver DNA sequences were present at the same place in the gel, i.e. within a restriction fragment of the same size. After reannealing, unhybridized single-stranded DNA was digested in situ with S1 nuclease. The hybridized tracer DNA was detected by autoradiography. The general applicability of this technique was demonstrated in the following experiments. The common EcoRI restriction fragments were identified in the genomes of E. coli and four other species of bacteria. Two of these fragments are conserved in all Enterobacteriaceae. In other experiments, repeated EcoRI fragments of eukaryotic DNA were visualized as bands of various intensity after reassociation of a total genomic restriction digest in the gel. The situation of gene amplification was modeled by the addition of varying amounts of lambda phage DNA to eukaryotic DNA prior to restriction enzyme digestion. Restriction fragments of lambda DNA were detectable at a ratio of 15 copies per chicken genome and 30 copies per human genome. This approach was used to detect amplified DNA fragments in methotrexate (MTX)-resistant mouse cells and to identify commonly amplified fragments in two independently derived MTX-resistant lines.

New physical map of bacteriophage T5 DNA.

The locations of 103 cleavage sites, produced by 13 restriction endonucleases, were mapped on the DNA of bacteriophage T5. Single- and double-digest fragment sizes were determined by agarose gel electrophoresis, using restriction fragments of phi X174 DNA and lambda DNA as molecular weight standards. Map coordinates were determined by a computer-based least-squares procedures (J. Schroeder and F. Blattner, Gene [Amst] 4:167-174, 1978). The fragment sizes predicted by the final map are all within 2% of the measured values. Based on this analysis, T5st(+) DNA contains 121,300 base pairs (Mr, 80.3 X 10(6) and has a terminal repetition of 10,160 base pairs (Mr, 6.7 X 10(6)). Restriction endonuclease analysis after treatment with exonuclease III and a single-strand-specific endonuclease allowed precise localization of five of the natural single-chain interruptions in T5 DNA. Revised locations for several T5 deletions were also determined.

Mechanism of degradation of duplex DNA by the DNase induced by herpes simplex virus.

Reaction intermediates formed during the degradation of linear PM2, T5, and lambda DNA by herpes simplex virus (HSV) DNase have been examined by agarose gel electrophoresis. Digestion of T5 DNA by HSV type 2 (HSV-2) DNase in the presence of Mn(2+) (endonuclease only) gave rise to 6 major and 12 minor fragments. Some of the fragments produced correspond to those observed after cleavage of T5 DNA by the single-strand-specific S1 nuclease, indicating that the HSV DNase rapidly cleaves opposite a nick or gap in a duplex DNA molecule. In contrast, HSV DNase did not produce distinct fragments upon digestion of linear PM2 or lambda DNA, which do not contain nicks. In the presence of Mg(2+), when both endonuclease and exonuclease activities of the HSV DNase occur, most of the same distinct fragments from digestion of T5 DNA were observed. However, these fragments were then further degraded preferentially from the ends, presumably by the action of the exonuclease activity. Unit-length lambda DNA, EcoRI restriction fragments of lambda DNA, and linear PM2 DNA were also degraded from the ends by HSV DNase in the same manner. Previous studies have suggested that the HSV exonuclease degrades in the 3' --> 5' direction. If this is correct, and since only 5'-monophosphate nucleosides are produced, then HSV DNase should "activate" DNA for DNA polymerase. However, unlike pancreatic DNase I, neither HSV-1 nor HSV-2 DNase, in the presence of Mg(2+) or Mn(2+), activated calf thymus DNA for HSV DNA polymerase. This suggests that HSV DNase degrades both strands of a linear double-stranded DNA molecule from the same end at about the same rate. That is, HSV DNase is apparently capable of degrading DNA strands in the 3' --> 5' direction as well as in the 5' --> 3' direction, yielding progressively smaller double-stranded molecules with flush ends. Except with minor differences, HSV-1 and HSV-2 DNases act in a similar manner.

Sedimentation velocity of DNA in isokinetic sucrose gradients: calibration against molecular weight using fragments of defined length.

The relationship between sedimentation coefficient and molecular weight for DNA sedimenting in preformed alkaline and neutral sucrose gradients was determined using absolute molecular weight standards (restriction fragments of plasmid pBR322 and phage lambda DNA). The range of calibration for alkaline gradients was extended to small DNA fragments (652 base-pairs) for the first time. The exponent b in the equation S20 degrees, w = aMb was found to be 0.380 in neutral gradients and 0.410 in alkali. The latter value differs significantly from previous estimates. The gradients were isokinetic, and the distance sedimented was shown to be directly proportional to the sedimentation coefficient at all times.

A study of the reversibility of helix-coil transition in DNA.

The reversibility of DNA melting has been thoroughly investigated at different ionic strengths. We concentrated on those stages of the process that do not involve a complete separation of the strands of the double helix. The differential melting curves of pBR 322 DNA and a fragment of T7 phage DNA in a buffer containing 0.02M Na+ have been shown to differ substantially from the differential curves of renaturation. Electron-microscopic mapping of pBR 322 DNA at different degrees of unwinding (by a previously elaborated technique) has shown that the irreversibility of melting under real experimental conditions is connected with the stage of forming new helical regions during renaturation. In a buffer containing 0.2M Na+ the melting curves of the DNAs used (pBR322, a fragment of T7 phage DNA, a fragment of phage Lambda DNA, a fragment of phiX174 phage DNA) coincide with the renaturation curves, i.e. the process is equilibrium. We have carried out a semi-quantitative analysis of the emergence of irreversibility in the melting of a double helix. The problem of comparing theoretical and experimental melting curves is discussed.

Cleavage and methylation of DNA by the restriction endonuclease HinfIII isolated from Haemophilus influenzae Rf

HinfIII is a restriction enzyme isolated from Haemophilus influenzae strain Rf. It requires ATP for cleavage and S-adenosyl-methionine for methylation of DNA. This enzyme can be present in two forms: one with AdoMet † † Abbreviation used: AdoMet, S-adenosyl-methionine. bound to it, and a second form free of this cofactor. In the presence of AdoMet and ATP the enzyme cleaves ColE1 DNA molecules once, to produce unit-length linear molecules. The HinfIII endonuclease cleaves at unique sites, though not every site on every molecule is cut. The five HinfIII cleavage sites were mapped relative to the EcoRI restriction endonuclease cleavage site. If AdoMet is omitted from the enzyme reaction mixture, the second form of HinfIII enzyme cleaves ColE1 DNA into several fragments. An average of 6.2 ± 1 methyl groups are transferred to ColE1 DNA from AdoMet. The methyl groups were mapped relative to the HaeIII restriction endonuclease fragments. The position of methylation sites correlates well with the cleavage sites. The restriction activity of the HinfIII enzyme shows some dependence upon the structure of the substrate DNA. The linear molecule of ColE1 DNA is a poorer substrate than the supercoiled molecules. Lambda DNA fragments with molecular weights smaller than approximately 2 × 10 6 are not cleaved by HinfIII enzyme, but since they can be methylated the enzyme is able to recognize the specific sequences on these fragments.

A novel ColEl::Tn3 plasmid vector that allows direct selection of hybrid clones in E. coli

A plasmid, named pKY2289, consists of a whole ColE1 DNA molecule and a complete ampicillin transposon (Tn3). When induced, E. coli K-12 cells which carry pKY2289 promote synthesis of colicin E1, but they are not immune to colicin E1. Inserting a DNA fragment into the Eco RI or the XmaI site of this plasmid abolishes its ability to produce active colicin E1. Thus, cells carrying one of these in vitro recombinant pKY2289 plasmids are able to form normal colonies in the presence of 0.1 μg/ml of mitomycin C and 50 μg/ml of ampicillin, while cells carrying the parental pKY2289 form very tiny colonies under the same conditions. This allows a positive selection for an in vitro recombinant pKY2289 molecule carrying a foreign DNA insertion. The properties of cells carrying the original pKY2289 are described and its potential usefulness as a cloning vehicle is demonstrated by cloning all the EcoRI and XmaI fragments of lambda DNA.

In vivo and in vitro recombination of lambda DNA in CaC12 transfection.

Using CaCl2 mediated transfection with Lambda DNA fragments, in vitro joining by ligase and in vivo recombination with helper phage DNA are effective systems for generating artificial recombinants. Recombination efficiencies are 20--30% in the in vitro and in vivo recombination systems. At 30 to 37 degree C T4 ligase mainly joins natural cohesive alpha ends, while at 12 degrees C the EcoRI-generated termini are preferentially ligated to form biologically active molecules, if the cloning vector alpha 401 is used, which has only one EcoRI target. The ligation products were characterized by gel electrophoresis and CaCl2 transfection. For in vivo recombination a new CaCl2 transfection system was developed, termed postinfection-dependent CaCl2 transfection system, which is based on the infection of recipient cells with helper phages after transfection. In marker rescue experiments using this method not only single but also double recombination occurred between two independent alpha DNA fragments and the helper phage DNA.