Showing papers on "In vitro recombination published in 1983"
TL;DR: A sensitive and general technique has been devised for the dual purposes of cloning genes by using antibodies as probes and isolating unknown proteins encoded by cloned DNA using an expression vector that permits insertion of foreign DNA into the beta-galactosidase structural gene lacZ and promotes synthesis of hybrid proteins.
Abstract: A sensitive and general technique has been devised for the dual purposes of cloning genes by using antibodies as probes and isolating unknown proteins encoded by cloned DNA. The method uses an expression vector, lambda gt11 (lac5 nin5 cI857 S100), that permits insertion of foreign DNA into the beta-galactosidase structural gene lacZ and promotes synthesis of hybrid proteins. Efficient screening of antigen-producing clones in lambda gt11 recombinant cDNA libraries is achieved through lysogeny of the phage library in hflA (high-frequency lysogeny) mutant cells of Escherichia coli; lysogens produce detectable quantities of antigen on induction, even when plated at high cell densities. The vector is also designed to facilitate the isolation of proteins specified by previously cloned gene sequences. Hybrid proteins encoded by recombinant phage accumulate in strains defective in protein degradation (lon mutants) in amounts amenable to large-scale purification. Antibodies produced against the portion of the hybrid encoded by foreign DNA could in turn be used to isolate the native polypeptide from eukaryotic cells.
1,998 citations
TL;DR: These vectors have been used successfully for DNA sequencing with the dideoxy-method, and can be used for any other purpose for which M13 derivatives are used, however, the pEMBL plasmids have the advantage of being smaller than M13 vectors, and the purification of the DNA is simpler.
Abstract: We have constructed a series of plasmids, the pEMBL family, characterized by the presence of 1) the bla gene as selectable marker, 2) a short segment coding for the alpha-peptide of beta-galactosidase and containing a multiple cloning sites polylinker, 3) the intragenic region of phage F1. pEMBL plasmids have the property of being encapsidated as single stranded DNA, upon superinfection with phage F1. These vectors have been used successfully for DNA sequencing with the dideoxy-method, and can be used for any other purpose for which M13 derivatives are used. However, the pEMBL plasmids have the advantage of being smaller than M13 vectors, and the purification of the DNA is simpler. In addition, and most importantly, long inserts have a higher stability in pEMBL plasmids than M13 vectors.
1,049 citations
TL;DR: This chapter describes one such method that uses an alkaline extraction step, rapid enough to be used as a screening method, permitting 50-100 or more samples to be extracted in a few hours, and sufficiently pure to be digestible by restriction enzymes, an important advantage for screening.
Abstract: Publisher Summary Plasmids are double-stranded circular DNA molecules that have the property of self-replication, independent of chromosomal DNA. In bacteria, they carry genes that specify a variety of host properties. In recent years, naturally occurring plasmids have been modified to produce new plasmids, which are used as cloning vehicles in recombinant DNA research. Although the presence of a plasmid in a bacterial cell may be detected genetically as a change in phenotype (e.g., resistance to a particular antibiotic), often it is necessary to isolate plasmid DNA for molecular studies, such as size determination, restriction enzyme mapping, and nucleotide sequencing, or for the construction of new hybrid plasmids. The degree of purification required depends upon the intended use. Highly purified material can be prepared by the "cleared lysate" method, which involves a long period of centrifugation in a dye-CsCl gradient. Less purified plasmid DNA is often satisfactory for recombinant DNA studies, and a large number of shorter and simpler methods have been developed. This chapter describes one such method that uses an alkaline extraction step. It is rapid enough to be used as a screening method, permitting 50-100 or more samples to be extracted in a few hours. The DNA is sufficiently pure to be digestible by restriction enzymes, an important advantage for screening. A preparative version that allows isolation of larger quantities of more highly purified material is also described.
1,027 citations
TL;DR: A Ti plasmid mutant was constructed in which all the on‐cogenic functions of the T‐DNA have been deleted and replaced by pBR322 and is proposed as an extremely versatile vector for the introduction of any DNA of interest into plant cells.
Abstract: A Ti plasmid mutant was constructed in which all the on-cogenic functions of the T-DNA have been deleted and replaced by pBR322 This Ti plasmid, pGV3850, still mediates efficient transfer and stabilization of its truncated T-DNA into infected plant cells Moreover, integration and expression of this minimal T-DNA in plant cells does not interfere with normal plant cell differentiation A DNA fragment cloned in a pBR vector can be inserted in the pGV3850 T-region upon a single recombination event through the pBR322 region of pGV3850 producing a co-integrate useful for the transformation of plant cells Based upon these properties, pGV3850 is proposed as an extremely versatile vector for the introduction of any DNA of interest into plant cells
800 citations
TL;DR: Analysis of substrate specificity with synthetic DNAs and restriction fragments of phi X174 replicative form DNA and pBR322 DNA showed that neither enzyme had dependence on the sequence context of CpG dinucleotides; poly(dG-dC) had the greatest methyl- accepting activity of any unmethylated DNA substrate tested.
Abstract: Dye-ligand chromatography on Cibacron blue F3GA-agarose has been used to resolve two species of DNA (cytosine-5-)-methyltransferase from nuclear extracts of uninduced Friend murine erythroleukemia cells. Each species has been highly purified; the activities in the first and second peaks were associated with polypeptides of Mr 150,000 and 175,000, respectively. Analysis of substrate specificity with synthetic DNAs and restriction fragments of phi X174 replicative form DNA and pBR322 DNA showed that neither enzyme had dependence on the sequence context of CpG dinucleotides; poly(dG-dC) had the greatest methyl-accepting activity of any unmethylated DNA substrate tested. De novo methylation by both enzymes was inefficient relative to methylation of hemimethylated sites. Methyl-accepting activity was strongly dependent on DNA chain length. This observation suggests that binding to DNA, followed by one-dimensional diffusion of enzyme along the DNA molecule, is important in the mechanism by which DNA methyltransferase locates its recognition sites.
367 citations
TL;DR: The ability to produce free products starting with a supercoiled substrate suggests a rather unique property of Cre-mediated lox recombination, the implications of which are discussed in terms of possible effects of the protein on the topology of the DNA molecule.
350 citations
TL;DR: To test the capacity of poxviruses for added foreign DNA, a recombinant was constructed that contains 24 700 bp of bacteriophage lambda DNA inserted within the vaccinia virus thymidine kinase (TK) gene.
317 citations
TL;DR: In this article, the authors present an important step towards solving the problem by way of genetic engineering, in that it provides recombinant DNA containing a DNA sequence occurring in the human factor IX genome.
Abstract: It has been a problem to find an alternative, less time-consuming, and more reliable source of factor IX, a polypeptide which is essential to the human blood-clotting process and necessary for the treatment of patients with Christmas disease The invention is an important step towards solving the problem by way of genetic engineering, in that it provides recombinant DNA containing a DNA sequence occurring in the human factor IX genome It includes recombinant DNA comprising substantially the whole sequence of human factor IX genome inserted in a cloning vehicle and transformed into a host such as Ecoli It is conveniently characterised by a 129 or 203- nucleotide long sequence (J-J' and J'-J'' in Figure 9) Other fragments of the sequence have also been cloned and the invention includes DNA molecules comprising part or all of the human factor IX DNA The invention also includes cDNA derived from human factor IX RNA Uses of the invention include the provision of an intermediate of value in the genetic engineering of a factor IX polypeptide precursor and thence manufacture of the factor IX polypeptide, and in making probes for use in diagnosing the presence of normal or abnormal factor IX DNA in patients with Christmas disease
298 citations
TL;DR: The lacI gene of Escherichia coli was used to score mutation in mammalian cells of simian virus 40-based recombinant DNA vectors that provide for replication and selection in both bacterial and mammalian cells.
Abstract: The lacI gene of Escherichia coli was used to score mutation in mammalian cells of simian virus 40-based recombinant DNA vectors that provide for replication and selection in both bacterial and mammalian cells. Plasmid DNA was introduced into COS7 simian cells by DEAE-dextran transfection, allowed to replicate in the mammalian cells, and then returned to E. coli for analysis. Mutants in lacI were observed at frequencies of one to several percent, compared with a spontaneous mutation rate in E. coli of less than 10(-5). The lesions include a large number of base substitutions, in addition to deletions, duplications, and more complex rearrangements, including insertion into the plasmid of sequences originating in the host genome. We discuss possible sources of the high mutation frequency and its implications for experiments involving DNA transfer.
288 citations
TL;DR: Mutants of Autographa californica and Galleria mellonella nuclear polyhedrosis viruses, which produce an altered plaque phenotype as a result of reduced numbers of viral occlusions in infected cells, were isolated after passage in Trichoplusia ni (TN-368) cells and it was suggested that the insertion of cell DNA into the viral genomes resulted in the FP plaque phenotype.
Abstract: Mutants of Autographa californica and Galleria mellonella nuclear polyhedrosis viruses, which produce an altered plaque phenotype as a result of reduced numbers of viral occlusions in infected cells, were isolated after passage in Trichoplusia ni (TN-368) cells. These mutants, termed FP (few-polyhedra) mutants, had acquired cell DNA sequences ranging from 0.8 to 2.8 kilobase pairs in size. The insertions of cell DNA occurred in a specific region between 35.0 and 37.7 map units of the A. californica viral genome. A cloned viral fragment containing one of the host DNA inserts was homologous to host DNA inserts in two other mutant viruses and to dispersed, repetitious sequences in T. ni cell DNA. Most of the homology between the cloned insert and cell DNA was contained within a 1,280-base-pair AluI fragment. Marker rescue studies and analysis of infected-cell-specific proteins suggested that the insertion of cell DNA into the viral genomes resulted in the FP plaque phenotype, possibly through the inactivation of a 25,000-molecular-weight protein.
281 citations
TL;DR: New phage lambda-based cloning vectors, Charons 32, 33, 34 and 35, have been constructed that allow cloning of large (19-21 kb) DNA fragments in up to six cloning sites.
TL;DR: Transformation in Synechococcus R2 can be exploited to modify precisely and extensively the genome of this photosynthetic microorganism.
TL;DR: The transposition-replication reaction of phage Mu has been reproduced in a cell-free reaction system and both types of products show incorporation of radioactive DNA precursors; however, simple inserts do not seem to undergo a full round of DNA replication.
TL;DR: The developed phage λ chromosome as a receptor for fragments of DNA was assured by the availability of well-characterized deletion mutants, by the means of selecting for the loss of restriction targets and by prior identification of the essential components of the phage transcriptional circuits and replication system.
Abstract: For more than 20 years, transducing derivatives of lambdoid phages have facilitated functional and structural analyses of bacterial genes. Restriction enzymes that make staggered cuts within specific DNA sequences (targets) produce discrete fragments with short cohesive ends (Hedgpeth et al. 1972; Mertz and Davis 1972; Bigger et al. 1973), and this discovery immediately indicated that fragments of DNA could be spliced into the severed arms of a λ vector molecule. The cohered fragments would then be joined covalently by DNA ligase (Gellert 1967), and the recombinant genomes, transducing phages, recovered by transfection of Escherichia coli (Mandel and Higa 1970). This goal was quickly realized (Murray and Murray 1974; Thomas et al. 1974), and detailed analyses of the type previously possible for only a few E. coli genes became generally applicable. The ability to insert DNA fragments from any source into plasmid and phage vectors, supplemented with the new techniques for rapid DNA sequence determination (Maxam and Gilbert 1977; Sanger et al. 1977), has already generated many exciting advances in diverse areas of molecular biology. The development of the phage λ chromosome as a receptor for fragments of DNA was assured by the availability of well-characterized deletion mutants, by the means of selecting for the loss of restriction targets and by prior identification of the essential components of the phage transcriptional circuits and replication system. The precise knowledge attained by 1972 showed the genetic engineer which regions of the λ genome could be sacrificed and, also, which special features could be...
TL;DR: Two large open reading frames were identified in the S-2 DNA, suggesting the possibility of protein-encoding genes, and the complete nucleotide sequence was determined by the chain termination method.
Abstract: Mitochondria from the S male-sterile cytoplasm (cms-S) of maize contain two plasmid-like DNAs, S-1 and S-2, that appear to be prominently involved with the cytoplasmic male sterility trait. The complete nucleotide sequence of the S-2 DNA molecule was determined by the chain termination method. The linear S-2 DNA molecule contains 5,452 base pairs and is terminated by exact 208-base-pair inverted repetitions. Two large open reading frames were identified in the S-2 DNA, suggesting the possibility of protein-encoding genes. The nucleotide sequence of the S-2 termini are discussed with regard to models proposed for the replication of linear DNA molecules.
TL;DR: This chapter explains the interaction of T4 topoisomerase with DNA in the origin region of the T4 chromosome and attempts to reconstitute the initiation reaction in vitro in order to test this and other models for the involvement of the enzyme in the DNA replication process.
Abstract: Publisher Summary Topoisomerases are enzymes that alter DNA topology by changing the linking number of circular duplex DNA molecules and by interconverting topologically knotted or catenated DNA forms. The so-called type II topoisomerases act by a mechanism involving the passage of a duplex segment of DNA through a transient double-strand break in another segment of DNA. A novel ATP-dependent type II topoisomerase with DNA-dependent ATPase activity is isolated from extracts of bacteriophage T4-infected E. coli cells. It has a high specific activity for topoisomerization reactions and can be easily purified to near homogeneity in milligram amounts. This chapter explains the interaction of T4 topoisomerase with DNA in the origin region of the T4 chromosome and attempts to reconstitute the initiation reaction in vitro in order to test this and other models for the involvement of the enzyme in the DNA replication process.
TL;DR: The origin of plasmid DNA transfer, oriT, has been localized on RK2, a conjugative drug-resistance plasmids of the IncP group with a very broad host range in gram-negative bacteria.
Abstract: The origin of plasmid DNA transfer, oriT, has been localized on RK2, a conjugative drug-resistance plasmid of the IncP group with a very broad host range in gram-negative bacteria. The transfer origin is contained in a 760-base-pair Hae II restriction fragment that maps in the same region as the single-strand nick made by the RK2 relaxation complex. The functional oriT was subcloned as a 112-base-pair Hpa II fragment, and the DNA sequence of this region was determined. The dominant structural feature of the oriT sequence is a 19-base-pair inverted repeat, with 15 of the 19 bases able to form pairs in a hairpin structure. This inverted repeat may be the recognition site for the relaxation complex proteins, which nick the plasmid DNA molecule and initiate the transfer process.
TL;DR: A strategy for kilo-base sequencing of a target DNA cloned in plasmid pWR34, where both strands of any DNA several thousand base pairs in length can be completely sequenced with ease within a short time, without the need for constructing a physical map.
Abstract: A strategy for kilo-base sequencing of a target DNA cloned in plasmid pWR34 is described. A long target DNA is progressively shortened from one end, by digestion with BAL31 nuclease or exonuclease III and nuclease S1, followed by cleaving off the shortened vector DNA. The family of the shortened target DNA molecule is next cloned in between the StuI site on one end, and a cohesive-ended restriction site on the other end, within the polylinker region of pWR34. DNA fragments cloned into this plasmid are sequenced directly by using a synthetic oligonucleotide primer, which binds to one side of the polylinker region using the dideoxynucleotide chain-termination method. The plasmid DNA, easily obtained by adoption of a rapid mini-preparation, is usually pure enough for direct DNA sequencing. Thus, both strands of any DNA several thousand base pairs in length can be completely sequenced (using two different primers) with ease within a short time, without the need for constructing a physical map.
TL;DR: This work demonstrates that FLP protein is actively involved in 2-microns plasmid recombination, and no other factors specific to yeast are required for the reaction and FLP-promoted recombination is not dependent upon any DNA structural features unique to yeast chromatin.
Abstract: The FLP gene of the yeast 2-microns plasmid is involved in a site-specific recombination event that results in the inversion of a set of sequences within the plasmid. This gene has been cloned and expressed in Escherichia coli. Expression of the FLP gene results in efficient recombination within the bacterial cell, which is specific for plasmids containing at least one 2-microns plasmid recombination site. This work demonstrates that (i) FLP protein is actively involved in 2-microns plasmid recombination; (ii) no other factors specific to yeast are required for the reaction; (iii) FLP protein acts efficiently in trans; (iv) FLP protein will promote site-specific insertion and deletion reactions in addition to the inversion reaction; and (v) FLP-promoted recombination is not dependent upon any DNA structural features unique to yeast chromatin.
TL;DR: The bacteriophage T4 helix-destabilizing protein, the product of gene 32, has been immobilized on an agarose matrix and used for affinity chromatography of lysates of T4-infected Escherichia coli cells.
Abstract: The bacteriophage T4 helix-destabilizing protein, the product of gene 32, has been immobilized on an agarose matrix and used for affinity chromatography of lysates of T4-infected Escherichia coli cells. At least 10 T4-encoded early proteins and 3 or 4 host proteins are specifically retained by this gene 32 protein column. Nine of the T4 proteins have been identified as being involved in either DNA replication or genetic recombination. Notably, the T4 DNA polymerase (gene 43 protein) and two major proteins in the recombination pathway (the products of genes uvsX and uvsY) are specifically bound. On a preparative scale, the column is useful for purification of the bound proteins.
TL;DR: In this article, two linear double-stranded DNA plasmids, k1 and k2, were used as a vector to transform a uracil-requiring strain of K. lactis (uraA mutant) to a prototrophic form.
Abstract: Some strains of Kluyveromyces lactis contain two linear double-stranded DNA plasmids, k1 and k2. The presence of the two plasmids confer on the cell a "killer" character, due to the production of a toxin that kills the sensitive cells. We have used one of these linear DNA molecules as a gene vector to transform K. lactis cells. Hybrid plasmids containing parts of the k1 plasmid and the URA3 gene of Saccharomyces cerevisiae have been constructed. We have found that the hybrid plasmids were able to transform a uracil-requiring strain of K. lactis (uraA mutant) to a prototrophic form. The transformed phenotype cosegregated with the hybrid plasmids. The transforming plasmids contained the sequence of one or both ends of the linear k1 DNA, but they were integrated into a circular molecule.
TL;DR: It is demonstrated that donor DNA enters Haemophilus influenzae through specialized membranous extensions, which are termed "transformasomes," which are in a protected state--resistant to external DNase and cellular restriction enzymes, although remaining unmodified and double-stranded.
Abstract: The mechanism by which Haemophilus protects donor DNA from cellular restriction and degradative enzymes during transformation is unclear. In this report, we demonstrate that donor DNA enters Haemophilus influenzae through specialized membranous extensions, which we have termed "transformasomes." DNA within transformasomes is in a protected state--resistant to external DNase and cellular restriction enzymes, although remaining unmodified and double-stranded. The ability of donor DNA to exit from transformasomes is dependent on its topological conformation. Circular DNA remains intact within transformasomes, while linear DNA rapidly exits and undergoes homologous recombination. Protected donor DNA can be preferentially removed from the surface of competent cells by extraction with organic solvents. Structurally intact transformasomes containing donor DNA could be partitioned into the organic layer and can be further purified by density centrifugation.
TL;DR: Linear double-stranded (ds)DNA, obtained by excision of the cloned A and B components of tomato golden mosaic virus (TGMV) from recombinant plasmids, was found to infect plants and to elicit symptoms identical to those obtained with T GMV or TGMV DNA.
Abstract: Linear double-stranded (ds)DNA, obtained by excision of the cloned A and B components of tomato golden mosaic virus (TGMV) from recombinant plasmids, was found to infect plants and to elicit symptoms identical to those obtained with TGMV or TGMV DNA. Progeny virus isolated from plants infected with cloned DNA was infective and indistinguishable from TGMV on the basis of (a) its circular single-stranded (ss)DNA genome, (b) its capsid polypeptide, (c) its particle morphology and (d) serological identity. Southern blot analysis of DNA extracted from cells infected with cloned DNA, or TGMV DNA, revealed the same intracellular ss and dsDNA species, represented in both A and B components, except for a subgenomic, possibly defective, DNA, which was not detected in infections with cloned DNA. Infection with cloned DNA was achieved when cloned A and B components were both present, but not with either cloned A or B components separately. TGMV is the first DNA virus for which unequivocal proof of a bipartite genome has been obtained.
TL;DR: 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.
Abstract: 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.
Patent•
10 Aug 1983TL;DR: In this paper, the authors present novel DNA sequences operative as promoters of microbial transcription of structural genes and comprising synthetic E.coli bacteriophage T5 "early" promoter replica DNA sequences.
Abstract: Disclosed are novel DNA sequences operative as promoters of microbial transcription of structural genes and comprising synthetic E.coli bacteriophage T5 "early" promoter replica DNA sequences. Disclosed also are novel promoter/operator DNA sequences comprising a synthetic T5 early promoter replica DNA sequence associated with a regulatable operator DNA sequence providing for selectively regulatable transcription of structural genes in, e.g., E.coli cells transformed with a DNA vector including same.
TL;DR: DNA-mediated gene transfer (transfection) is used to introduce specific genes into vertebrate cells by determining the infectivity of the DNA from an avian retrovirus and of mixtures of subgenomic fragments of this DNA.
Abstract: DNA-mediated gene transfer (transfection) is used to introduce specific genes into vertebrate cells. Events soon after transfection were quantitatively analyzed by determining the infectivity of the DNA from an avian retrovirus and of mixtures of subgenomic fragments of this DNA. The limiting step of transfection with two DNA molecules is the uptake by a single cell of both DNA's in a biologically active state. Transfected cells mediate ligation and recombination of physically unlinked DNA's at nearly 100 percent efficiency.
TL;DR: Analysis of three integrated HBV DNA sequences cloned from DNA of the human PLC cell line PLC/PRF/5 shows no obvious rearrangements in these clones, but integration can be associated with deletions in the viral DNA and also apparently in the host DNA.
TL;DR: An improved procedure for the efficient addition of homopolymer tails to DNA is presented and a new procedure forThe addition of a single nucleotide to the 3´ end of a DNA is described as a method for in vitro mutagenesis, which can prove to be useful in investigating the control and expression of genes.
Abstract: Publisher Summary Terminal deoxynucleotidyltransferase catalyzes the addition of deoxynucleotides to the 3´ termini of DNA. In 1962, this enzyme was recognized as being different from DNA polymerase, and the name was changed to terminal transferase. By use of a tailing method, which adds homopolymer deoxynucleotide tails to denatured DNA, any double-stranded DNA fragment can be joined to a cloning vehicle. Because each DNA fragment carries the same type of tail, it cannot hybridize with another molecule of the same species. Thus, after cloning, each transformant should represent the desired recombinant DNA. These advantages make this technique particularly useful for the construction of recombinant DNA molecules for cloning. This chapter presents an improved procedure for the efficient addition of homopolymer tails to DNA, and also describes a new procedure for the addition of a single nucleotide to the 3´ end of a DNA as a method for in vitro mutagenesis, which can prove to be useful in investigating the control and expression of genes.
TL;DR: It is shown that the cruciform structure rarely if ever exists in vivo, but after DNA isolation greater than 90% of the sequence is in cruciforms and results suggest that the plasmid DNA as organized in vivo either lacks sufficient torsional tension to form this cruciform or the palindrome is restrained in the linear form by other bound molecules.
Abstract: A perfect palindromic 66-base pair (bp) DNA sequence derived from the lac operator and cloned into plasmid pMB9 [Betz, J. L. & Sadler, J. R. (1981) Gene 13, 1-12] can exist in a 66-bp linear form or as two 33-bp cruciform arms. The fraction of the sequence in the cruciform depends on the superhelical density of the plasmid DNA. Relaxed DNA contains no cruciforms. The palindrome in the cruciform structure is cut by EcoRI endonuclease at the base of the cruciform arms, releasing 33-bp fragments; when in the linear form only 66-bp fragments are produced. The cruciform structure is fixed by trimethylpsoralen crosslinks in the cruciform arms. This together with the EcoRI cutting provides an assay for the cruciform structures in the DNA of living cells. Using this assay we show that the cruciform structure rarely if ever exists in vivo, but after DNA isolation greater than 90% of the sequence is in cruciforms. Results suggest that the plasmid DNA as organized in vivo either lacks sufficient torsional tension to form this cruciform or the palindrome is restrained in the linear form by other bound molecules.
TL;DR: Observations suggest that signals required for the encapsidation of HSV-1 DNA are located within DNA sequences shared by the inserted fragments and therefore lie within the reiterated 'a' sequence of the viral genome.
Abstract: A 535 base pair DNA fragment which maps entirely within the IRS/TRS regions of the herpes simplex virus type 1 (HSV-1) genome and contains all the cis-acting signals necessary for it to function as an origin of viral DNA replication has previously been identified (N.D. Stow and E.C. McMonagle, Virology, in press). When BHK cells were transfected with circular plasmid molecules containing cloned copies of this DNA fragment, and superinfected with wt HSV-1 as helper, amplification of the input plasmid was detected. Two observations indicated that the amplified DNA was not packaged into virus particles. Firstly, when the transfected cells were disrupted the amplified DNA was susceptible to digestion by added DNase, and secondly, it was not possible to further propagate the DNA when virus from the cells was passaged. Fragments from the joint region and from both termini of the viral genome were inserted into origin-containing plasmids and the resulting constructs analysed. In all cases the inserted fragment allowed the amplified DNA to be further passaged, and a proportion to become resistant to digestion with DNase. These observations suggest that signals required for the encapsidation of HSV-1 DNA are located within DNA sequences shared by the inserted fragments and therefore lie within the reiterated 'a' sequence of the viral genome.