Showing papers on "Base pair published in 1972"
TL;DR: Methods for covalently joining duplex DNA molecules to one another are developed and used to construct circular dimers of SV40 DNA and to insert a DNA segment containing lambda phage genes and the galactose operon of E. coli into SV40DNA.
Abstract: We have developed methods for covalently joining duplex DNA molecules to one another and have used these techniques to construct circular dimers of SV40 DNA and to insert a DNA segment containing lambda phage genes and the galactose operon of E coli into SV40 DNA The method involves: (a) converting circular SV40 DNA to a linear form, (b) adding single-stranded homodeoxypolymeric extensions of defined composition and length to the 3′ ends of one of the DNA strands with the enzyme terminal deoxynucleotidyl transferase (c) adding complementary homodeoxypolymeric extensions to the other DNA strand, (d) annealing the two DNA molecules to form a circular duplex structure, and (e) filling the gaps and sealing nicks in this structure with E coli DNA polymerase and DNA ligase to form a covalently closed-circular DNA molecule
673 citations
TL;DR: The gradual bimodal decrease in sedimentation rate seen after amino-acid starvation suggests that the faster sedimenting structures in exponentially growing cells represent the folded chromosomes at more advanced stages in their replication cycle.
587 citations
TL;DR: Some potential applications of these analogues in studies of the structure and function of nucleic acids and proteins are: analysis of protein-nucleic acid interaction, polynucleotide conformation and conformational transitions, enzyme-coenzyme interaction, and nuclease assay.
575 citations
TL;DR: It is concluded that RNA polymerase can exist in two conformational states, one programmed for promoter binding and initiation (holoenzyme) and the other designed for RNA chain elongation (core polymerase).
416 citations
TL;DR: It is hypothesized that the leukovirus RNA-directed DNA polymerase activity is an integral part of the ribonucleoprotein core of the virions, which suggests that the virion enzyme activity is related to normal cellular DNA polymerases, and that there are homologies between the amino acid sequences of the viral enzyme and normal cellular enzymes.
Abstract: Publisher Summary The discovery of RNA-directed DNA synthesis in disrupted virions of RNA tumor viruses added strong support to the hypothesis that information transfer from RNA to DNA exists in biological system. The chapter discusses the properties of the endogenous reaction carried out by the virion DNA polymerase. To study the endogenous reaction disrupted, virions are incubated with substrates in the absence of any added template and synthesis of DNA is observed using the RNA present in the virions as template. The chapter also discusses the general implications of RNA-directed DNA synthesis in relation to tumor viruses, neoplastic cells, and normal cells. It is hypothesized that the leukovirus RNA-directed DNA polymerase activity is an integral part of the ribonucleoprotein core of the virions. The cores are synthesized in cells as precursor particles and then are incorporated into complete virions when the virions are assembled by budding at the cell surface. This core enzyme system contains not only the template-primer RNA, a DNA polymerase that can transfer information from RNA to double-stranded DNA, but ancillary enzymes, such as polynucleotide ligase and nucleases, which may aid in integrating the viral information with cellular DNA. This suggests that the virion enzyme activity is related to normal cellular DNA polymerases, and that there are homologies between the amino acid sequences of the viral enzyme and normal cellular enzymes. The relationship of RNA-directed DNA synthesis to neoplasia depends upon the relationship of RNA tumor viruses to neoplasia, which is supported by three general hypotheses: the provirus model, the oncogene model, and the protovirus model.
400 citations
TL;DR: The sequence of DNA base pairs adjacent to the phosphodiester bonds cleaved by the RI restriction endonuclease in unmodified DNA from coliphage lambda has been determined and its most striking feature is its symmetry.
Abstract: The sequence of DNA base pairs adjacent to the phosphodiester bonds cleaved by the RI restriction endonuclease in unmodified DNA from coliphage λ has been determined. The 5′-terminal nucleotide labeled with 32P and oligonucleotides up to the heptamer were analyzed from a pancreatic DNase digest. The following sequence of nucleotides adjacent to the RI break made in λ DNA was deduced from these data and from the 3′-dinucleotide sequence and nearest-neighbor analysis obtained from repair synthesis with the DNA polymerase of Rous sarcoma virus [Formula: see text] The RI endonuclease cleavage of the phosphodiester bonds (indicated by arrows) generates 5′-phosphoryls and short cohesive termini of four nucleotides, pApApTpT. The most striking feature of the sequence is its symmetry.
281 citations
TL;DR: It was demonstrated that actinomycin D at low concentration, 0.04 μg/ml, supresses the appearance of mRNA on polysomes to an extent of 50%.
Abstract: Messenger RNA from polysomes of KB-cells was isolated by affinity chromatography on columns of polyuridylic acid covalently linked to Sepharose. The mRNA molecules were retained by the resin apparently via their poly(A) segments by base pairing to the poly(U). Ribosomal RNA and transfer RNA were not retained by the columns and were thus removed from the mRNA. The mRNA was recovered to an extent of 90% and apparently in intact form.
This method allows studies of mRNA resulting from unabated synthesis and was used here in studies of the size distribution of different classes of cytoplasmic poly(A)-containing RNA. The presence of poly(A)-containing RNA in the non-polysomal fractions of the cytoplasm was demonstrated. This putative mRNA was shown to constitute about 30% of the total cytoplasmic poly(A)-containing RNA. Also by using the poly (U)-Sepharose technique it was demonstrated that actinomycin D at low concentration, 0.04 μg/ml, supresses the appearance of mRNA on polysomes to an extent of 50%. This low concentration of the drug was previously though to effect only ribosomal RNA synthesis.
207 citations
TL;DR: R(1) restriction endonuclease cleaves duplex DNA at a specific sequence, probably 6 nucleotide pairs in length, by making two single-strand staggered cleavages, generating 5'-phosphoryl and 3'-hydroxyl termini.
Abstract: R1 restriction endonuclease cleaves duplex DNA at a specific sequence, probably 6 nucleotide pairs in length, by making two single-strand staggered cleavages, generating 5′-phosphoryl and 3′-hydroxyl termini. The single-strand ends produced at each break have identical and complementary sequences of 4 or 6 nucleotides in length. Therefore, the cleavage site possesses a 2-fold rotational axis of symmetry perpendicular to the helix axis. The ends of full-length linear SV40 DNA, generated by R1 endonuclease cleavage, can be joined by Escherichia coli ligase to regenerate duplex, fully infectious, covalently-closed circular molecules. It was further found that all R1 endonuclease-generated ends are identical and complementary. Therefore, any two DNA molecules with R1 sites can be “recombined” at their restriction sites by the sequential action of R1 endonuclease and DNA ligase to generate hybrid DNA molecules.
201 citations
TL;DR: It was found that many insertions which often arise as strongly polar mutations consist of only a few “foreign” DNA sequences.
Abstract: It was found that many insertions which often arise as strongly polar mutations consist of only a few “foreign” DNA sequences. The most commonly observed “short” polar IS1 insertions are all comprised of the same DNA with a duplex length of 750±80 nucleotide pairs. This insertable IS1 DNA can be integrated with either orientation into the genome at various positions in the lac and gal operons of E. coli; however, the inserted sequence is not permuted. The r14 polar insertion in gene cII of phage λ also consists of the same IS1 DNA (Hirsch et al., 1972 b). “Long” insertions (1170 to 1490 nucleotide pairs) were detected in the lac (IS3) and gal (IS2, IS4) operons and in the y and P-Q regions (IS2) of the λ genome and measured as single-strand loops in the l/r heteroduplex DNA. The insertions in the latter two positions are homologous, although those found in the P-Q region do not exhibit any obvious polarity.
190 citations
TL;DR: Analyses of nascent nucleic acids from cells pulse labeled for various times, and of the molecules with different sizes, support the hypothesis that the short DNA fragments are formed by extension of even shorter RNA chains, which are synthesized on the parental DNA strands and are removed before ligation of the DNA fragments.
Abstract: Nucleic acid that is extracted from E. coli labeled by a brief pulse of [3H]dT and depatured by treatment with heat, formamide, or formaldehyde bands in a region with a density higher than that of single-stranded E. coli DNA in a Cs2SO4 equilibrium density gradient. If treated with alkali or RNase, it then exhibits the density of single-stranded DNA. These results suggest the presence of a short strand of RNA covalently linked to the nascent DNA. Evidence for the presence of covalently linked RNA-DNA molecules is also obtained by pulse labeling with [3H]U. Analyses of nascent nucleic acids from cells pulse labeled for various times, and of the molecules with different sizes, support the hypothesis that the short DNA fragments are formed by extension of even shorter RNA chains, which are synthesized on the parental DNA strands and are removed before ligation of the DNA fragments. The synthesis of the RNA segment of the RNA-DNA molecule is much less sensitive to rifampicin than is the synthesis of bulk RNA.
184 citations
TL;DR: Asymmetric RNA was synthesized in vitro from SV40 component I DNA using Escherichia coli DNA-dependent RNA polymerase and hybridized to RNA extracted at different stages of lytic infection and to RNA from transformed cells.
TL;DR: The data indicate the existence of a variable region of DNA with very little, if any, homology and an invariable region with relatively good matching of base pairs in herpes simplex virus.
Abstract: The extent of homology between herpes simplex virus(1) and(2) (HSV-1 and HSV-2) deoxyribonucleic acid (DNA) was measured in two ways: (i) by determination of the relative rate of hybridization of labeled HSV-1 and HSV-2 DNA to excess unlabeled HSV-1 or HSV-2 DNA immobilized on filters and (ii) by determination of the rate of hybridization of labeled HSV-1 and HSV-2 DNA to excess unlabeled HSV-1 or HSV-2 DNA in solution. Approximately 40% of HSV-1 and HSV-2 DNA is homologous at hybridization temperatures 25 C below the melting temperature (T(m)) of HSV DNA (liquid-filter annealing). Lowering the temperature to 34 C below the T(m) increased the extent of homology to 46% (liquid annealing). The extent of base-pairing in HSV-1-HSV-2 heteroduplex DNA was determined by thermal chromatography on hydroxyapatite. Heteroduplexes of HSV-1 and HSV-2 DNA eluted in a single peak whose midpoint (Te(50)) was 10 C below that of the homoduplex. Conspicuously absent were heteroduplexes that eluted at more than 15 C below the Te(50) of the homoduplex. The data indicate the existence of a variable region of DNA (54%) with very little, if any, homology and an invariable region (46%) with relatively good (85%) matching of base pairs.
TL;DR: The template requirements and deoxyribonucleic acid (DNA) products of the DNA polymerases isolated from Rauscher leukemia and avian myeloblastosis viruses have been examined and DNA preparations containing extensive single-stranded regions are poorly utilized as templates.
Abstract: The role of ribonucleic acid (RNA) in deoxyribonucleic acid (DNA) synthesis with the purified DNA polymerase from the avian myeloblastosis virus has been studied. The polymerase catalyzes the synthesis of DNA in the presence of four deoxynucleoside triphosphates, Mg(2+), and a variety of RNA templates including those isolated from avian myeloblastosis, Rous sarcoma, and Rauscher leukemia viruses; phages f2, MS2, and Qbeta; and synthetic homopolymers such as polyadenylate.polyuridylic acid. The enzyme does not initiate the synthesis of new chains but incorporates deoxynucleotides at 3' hydroxyl ends of primer strands. The product is an RNA.DNA hybrid in which the two polynucleotide components are covalently linked. Free DNA has not been detected among the products formed with the purified enzyme in vitro. The DNA synthesized with avian myeloblastosis virus RNA after alkaline hydrolysis has a sedimentation coefficient of 6 to 7S.
TL;DR: Four insertion sites in the omega region of gene z were identified and the position of the lac5 substitution and the SR2 deletion in the λplac DNA were determined by heteroduplex mapping.
Abstract: Several strongly polar mutations in the omega region of the z gene of the lac operon result from insertions consisting of only two specific sequences of DNA, one about 870 and the other 1170 nucleotide pairs long (based on single-strand measurements). No sequence homology was detected between the shorter (IS1) and longer (IS3) insertions. The IS1 insertion was shown to possess a specific attachment site, but it can be inserted with either orientation at several sites in the z gene. Four insertion sites in the omega region of gene z were identified and the position of the lac5 substitution and the SR2 deletion in the λplac DNA were determined by heteroduplex mapping.
TL;DR: It is concluded that the binding of RNA polymerase did not cause local unwinding but RNA synthesis produced "defects” in the double helix, and the interpretation of formaldehyde-induced denaturation is not clear, nor is the nature of a “defect”.
Abstract: IT has been a source of speculation whether the reading of the genetic code of DNA by RNA polymerase involves the disruption of the DNA helix. While circuitous evidence favouring either affirmative or negative answer has been accumulating, direct experiments have been few1–11. Kosaganov et al. investigated the possibility of a local unwinding of DNA during RNA synthesis by measuring the kinetics of formaldehyde-induced denaturation of DNA during RNA synthesis12. They concluded that the binding of RNA polymerase did not cause local unwinding but RNA synthesis produced “defects” in the double helix. Unfortunately, the interpretation of formaldehyde-induced denaturation is not clear, nor is the nature of a “defect”.
TL;DR: These data are supported by cross-hybridization experiments using RNA transcribed in vitro from lambda dgal or lambda DNA carrying one insertion and DNA carrying either the same or a different insertion.
Abstract: Six insertion mutations in the gal operon of E. coli and two insertion mutations in the xycIIOP operon of bacteriophage lambda were tested for homology by annealing separated strands of lambda dgal DNA carrying the insertions, and inspection in the electron microscope. Class 1, consisting of the gal mutations OP 128, OP 141, T-N 116, OP 306, T-N 102 and the lambda mutation r14 are about 800 nucleotide pairs long, completely homologous and not circularly permuted. The first three insertions of class 1 are integrated in one direction with respect to the adjacent genes, the other three in the opposite direction. The DNA inserted in this class of mutations is called IS1. Class 2 consists of the gal insertion OP 308 and the lambda insertion r32. They are about 1400 nucleotide pairs long. The two are integrated in opposite direction with respect to the chromosome of λdgal. The DNA in insertion mutations of class 2 will be called IS 2. IS1 and IS2 do not share any detectable homology. These data are supported by cross-hybridization experiments using RNA transcribed in vitro from lambda dgal or lambda DNA carrying one insertion and DNA carrying either the same or a different insertion. Similar results were obtained by Malamy, Fiandt, Szybalski and Fiandt, Szybalski, Malamy (accompanying papers).
TL;DR: Experiments are described which show that it is possible to deduce the analytical complexity of DNA with reasonable accuracy from its hybridization with complementary RNA and estimate the reiteration frequency of multiple DNA sequences from the hybridization of the total DNA with RNA complementary to the multiple sequences.
Abstract: When RNA is annealed in solution with a sufficiently large excess of DNA, the kinetics of DNA–RNA hybridization are relatively simple. Methods are described for following the course of both DNA renaturation and DNA–RNA hybridization in this system. To explore the characteristics of the reaction a series of model systems was used. Each one utilized DNA (sheared to constant size) from a bacterium or bacteriophage and homologous cRNA, i.e. RNA synthesized in vitro on a template of the same DNA. Temperature optima were determined for the hybridization of Escherichia coli nucleic acids in 2×SSC and 3×SSC-50% formamide buffers, and of Proteus mirabilis nucleic acids in 2×SSC buffer. Rate-constants for DNA–RNA hybridization were measured by two methods. These gave somewhat different results, but in all cases the rate-constant of DNA–RNA hybridization was clearly less than that of DNA renaturation. Thus hybridization is a slower reaction than DNA renaturation. Nevertheless, in some cases, with a high concentration of DNA and a long annealing time, 90–95% of the added RNA became resistant to ribonuclease. Experiments are described which show that it is possible to deduce the analytical complexity of DNA with reasonable accuracy from its hybridization with complementary RNA. Similarly, it is possible to estimate the reiteration frequency of multiple DNA sequences (such as ribosomal DNA) from the hybridization of the total DNA with RNA complementary to the multiple sequences. The effect on the system of various DNA/RNA ratios from 100 to 1 is described.
TL;DR: Unlike the case of mouse, human heterochromatin is composed of a variety of DNA families, and mapping human chromosomes by nucleic-acid hybridization appears feasible, the results suggest that repeated sequences in the DNA of the human genome suggest the heterogeneity of the repeated sequences.
TL;DR: The T4 ligase was found to bring about the joining reaction when the hydrogen-bonded duplexes contained mismatched (non-Watson-Crick) base pairs.
TL;DR: The circular dichroism of double-stranded DNA is temperature dependent prior to its melting, and it has been suggested previously that this pre-melt behavior represents a transition from B-DNA to A-DNA, but the data tend specifically to rule out this possibility.
TL;DR: Ritonucleic acid extracted from four stages during the developmental cycle of the cellular slime mold Dictyostelium discoideum was hybridized with purified single-copy nuclear DNA, indicating that there are, during development, qualitative and quantitative changes in the portion of the single- copies of the genome transcribed.
TL;DR: It is shown that protein synthesis is not required for the elimination of an inhibitor of DNA synthesis and no qualitative differences could be found between DNA synthesized in the absence of protein synthesis and DNA made in control cells.
TL;DR: The repetitive segments of the middle-repetitive portion of the DNA of Drosophila melanogaster have been found to be 150 base pairs or somewhat less in length.
TL;DR: In vitro DNA synthesis on single-stranded circular DNA can be initiated by RNA primers, and the possible role of ribonuclease H in RNA-primed DNA synthesis in vivo is discussed.
Abstract: In vitro DNA synthesis on single-stranded circular DNA can be initiated by RNA primers RNA chains are covalently extended by DNA polymerase II from KB cells and DNA polymerase I from Micrococcus luteus, but not by an RNA-dependent DNA polymerase from avian myeloblastosis virus The reaction product consists of DNA chains with a piece of RNA at their 5′-ends, hydrogen bonded to the template DNA The primer RNA is linked to the product DNA via a 3′:5′-phosphodiester bond, and can be specifically removed by ribonuclease H The possible role of ribonuclease H in RNA-primed DNA synthesis in vivo is discussed
TL;DR: The evidence from intersatellite reassociation and from pyrimidine tract analysis of the M. musctdus satellite suggests that the unstable satellite ancestor was itself a diverged repeating sequence, with a basic complexity of only 8 to 20 nucleotide pairs.
TL;DR: Conformationsl changes of DNA induced by netropsin binding have been studied by way of viscosity measurements at different DNA molecular weights on the basis of equations derived in the Appendix for semi-rigid polymers and specialized for DNA at standard conditions.
TL;DR: Selective methods for removing all but arginine-rich histones from calf thymus chromatin reveal that they are bound to DNA regions very rich in G·C base pairs.
Abstract: Selective methods for removing all but arginine-rich histones from calf thymus chromatin reveal that they are bound to DNA regions very rich in G·C base pairs.
TL;DR: Cytological hybridization experiments with RNA from two additional Balbiani rings indicated that the three BR's differ with regard to information content, and that BR-1 like BR-2 contains repeated nucleotide sequences, likely to be involved in the genetic determination of secretory proteins.
TL;DR: The DNA of bacteriophage T5 can be shown to be terminally repetitious by annealing after partial digestion with λ exonuclease, suggesting that a single-chain interruption is located within the terminal repetition.
TL;DR: The mechanism by which steroid-receptor complexes might regulate the transcription of the genome is yet to be determined, but it has been suggested that in the class of nuclear acidic proteins there are acceptor molecules which can bind specifically to the hormone- receptor complex and to DNA.
Abstract: STUDIES on the interactions of steroid hormones with their target tissues have led to the generally accepted idea that hormone-receptor complexes are in some way involved in altering the pattern of DNA transcription1–12. The mechanism by which steroid-receptor complexes might regulate the transcription of the genome is, however, yet to be determined. It has been suggested that in the class of nuclear acidic proteins there are acceptor molecules which can bind specifically to the hormone-receptor complex and to DNA13. Other studies have indicated that the integrity of DNA is important for the binding of the hormone-receptor complex in nuclei14,15. The result of the binding of steroid-receptor complexes to DNA could presumably be the activation of a specific set of genes.