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The Journal of Biological Chemistry Jul 1975In connection with work on the nucleotide sequence of the promoter for the gene N of bacteriophage lambda as well as a study of the mechanism of transcription, a 20-unit...
In connection with work on the nucleotide sequence of the promoter for the gene N of bacteriophage lambda as well as a study of the mechanism of transcription, a 20-unit long DNA duplex corresponding to the known sequence at the 5' end of the above gene transcript has been synthesized. For synthesis, the required duplex was divided into the following deoxyribooligonucleotides: a) the dodecanucleotide, d-A-T-C-A-G-C-A-G-G-A-C-G (II); b) the octanucleotide, d-C-A-C-T-G-A-C-C- (IV); c) the hexanucleotide, d-G-C-T-G-A-rU (I); and d) dodecanucleotide, d-T-C-A-G-T-G-C-G-T-C-C-T (III). All of the four olignucleotides were chemically synthesized and characterized by extensive chromatographic and fingerprinting methods (after labeling the 5' ends with[32P]phosphate group). Longer polynucleotides (an icosa- and an octadecanucleotide) were prepared by polynucleotide ligase-catalyzed joining of segments I and III and by joining segments II and IV. The use of the octadecanucleotide, d-T-C-A-G-T-G-C-G-T-C-C-T-G-C-T-G-A-rU, in work on the sequence analysis of the promoter is described in the accompanying paper. The octadecanucleotide and icosanucleotide were hybridized together to give the double-stranded duplex.
Topics: Adenosine Triphosphate; Autoradiography; Base Sequence; Chromatography, DEAE-Cellulose; Chromatography, Gel; Coliphages; DNA, Viral; Electrophoresis, Cellulose Acetate; Electrophoresis, Polyacrylamide Gel; Genes; Phosphorus Radioisotopes; Polynucleotide 5'-Hydroxyl-Kinase; Polynucleotide Ligases; Polynucleotides; RNA, Messenger; Transcription, Genetic
PubMed: 1141242
DOI: No ID Found -
Proceedings of the National Academy of... Sep 1967
Topics: Animals; Chemical Phenomena; Chemistry; Culture Techniques; Interferons; Mice; Polynucleotides; Rabbits; Trypsin; Virus Diseases
PubMed: 5233831
DOI: 10.1073/pnas.58.3.1004 -
Journal of Bacteriology Sep 1973Bacterial and plasmid homo- and heteroduplexes have been analyzed with a single-strand specific endonuclease, S1, of Aspergillus oryzae. Under appropriate assay...
Bacterial and plasmid homo- and heteroduplexes have been analyzed with a single-strand specific endonuclease, S1, of Aspergillus oryzae. Under appropriate assay conditions, there was a high degree of correlation between the degree of deoxyribonucleic acid (DNA)-DNA homoduplex formation assessed by the S1 endonuclease and by hydroxyapatite (HA). Heteroduplexes which contain extensive regions of polynucleotide sequences in common are similarly recognized by the S1 endonuclease and HA. In instances where there is little or imperfect complementarity between heterologous DNA strands, the S1 endonuclease and the HA method give slightly different estimates. From DNA duplex thermal stability experiments assayed with the S1 endonuclease, there is preliminary evidence that well-matched sequences identified by the enzyme are not similarly recognized by HA. The assay of homo- and heteroduplexes with the S1 endonuclease permits an accurate, reproducible and rapid determination of polynucleotide sequence relationships and may be seriously considered as a method of choice for survey work and for investigations which require a large number of DNA-DNA hybridization assays.
Topics: Aspergillus; Base Sequence; Carbon Isotopes; Coliphages; DNA, Bacterial; DNA, Single-Stranded; DNA, Viral; Deoxyribonucleases; Enterobacteriaceae; Hot Temperature; Hydroxyapatites; Methods; Nucleic Acid Denaturation; Nucleic Acid Hybridization; Phosphorus Isotopes; Polynucleotides; Species Specificity; Tritium
PubMed: 4728274
DOI: 10.1128/jb.115.3.904-911.1973 -
Journal of Biochemistry Jun 1964
Topics: DNA; DNA, Bacterial; Escherichia coli; Hot Temperature; Nitrites; Phosphorus Isotopes; Polydeoxyribonucleotides; Polynucleotides; RNA; RNA, Bacterial; Research; Ribonucleases; Ribosomes; Trypsin
PubMed: 14216417
DOI: 10.1093/oxfordjournals.jbchem.a127946 -
Nucleic Acids Research Jan 1998A simple and efficient method for in vitro mutagenesis and recombination of polynucleotide sequences is reported. The method involves priming template polynucleotide(s)...
A simple and efficient method for in vitro mutagenesis and recombination of polynucleotide sequences is reported. The method involves priming template polynucleotide(s) with random-sequence primers and extending to generate a pool of short DNA fragments which contain a controllable level of point mutations. The fragments are reassembled during cycles of denaturation, annealing and further enzyme-catalyzed DNA polymerization to produce a library of full-length sequences. Screening or selecting the expressed gene products leads to new variants with improved functions, as demonstrated by the recombination of genes encoding different thermostable subtilisins in order to obtain enzymes more stable than either parent.
Topics: Bacillus subtilis; DNA; DNA Polymerase I; DNA Primers; Enzyme Stability; Evolution, Molecular; Gene Expression; Mutagenesis; Point Mutation; Polynucleotides; Recombination, Genetic; Subtilisins; Templates, Genetic
PubMed: 9421535
DOI: 10.1093/nar/26.2.681 -
Nucleic Acids Research May 1987A method allowing routine establishment of full length and functionally competent cDNA clones of particular mRNAs from small preparations of polyadenylated RNA is...
A method allowing routine establishment of full length and functionally competent cDNA clones of particular mRNAs from small preparations of polyadenylated RNA is described. Pairs of synthetic primers are used for first and second strand synthesis. They include sequences complementary to the 3' terminal regions of the mRNAs and of the full length first cDNA strands, respectively and bear a few additional nucleotides at their 5' ends. After synthesis of both cDNA strands in one tube, they are precisely trimmed back with T4 DNA polymerase in presence of only two nucleoside triphosphates, to yield sticky ends fitting into a vector plasmid cleaved with two restriction endonucleases. The procedure was first applied to the simultaneous cloning of all five major measles virus (MV) mRNA species from a persistently infected cell line. Two thirds of all clones contained full length MV-specific cDNAs. Screening of less than 200 clones was sufficient to obtain several independent clones corresponding to each mRNA, except for gene F which was represented only once.
Topics: Cloning, Molecular; DNA; Measles virus; Poly A; Polynucleotides; RNA, Messenger; RNA, Viral
PubMed: 2884622
DOI: 10.1093/nar/15.10.3987 -
Biophysical Chemistry Apr 1975The hysteresis observed in cyclic acid-base titrations of the three-standed polyribonucleotide helix poly (A)-2 POLY (U) strongly depends on ionic strength. For NaCl and...
The hysteresis observed in cyclic acid-base titrations of the three-standed polyribonucleotide helix poly (A)-2 POLY (U) strongly depends on ionic strength. For NaCl and at 25 degrees C, hysteresis occurs in the limited concentration range between 0.03 M and 1.0 M(NaCl). The transition points associated with the cyclic conversions between the triple helix and the poly (A)-poly (A) double helix and (free) poly (U) constitute a (pH ionic strength) phase diagram covering the ranges of stability and metastability of the hysteresis system. Variations with NaCl concentration of some hysteresis parameters can be quantitatively described in terms of polyelectrolyte theories based on the cylinder-cell model for rodlike polyions. The results of this analysis suggest that the metastability is predominantly due to dlectrostatic energy barriers preventing the equilibrium transition of the partially protonated triple helix above a critical pH value. Ultraviolet absorbance and potentiometric titration data of poly (A)in the acidic pH range can be analyzed in terms of two types of double-helical structures. Spectrophotometric titrations reveal isosbestic wavelengths for structural transitions of poly (A). "Time effects" commonly observed in poly (A) titrations are suggested to reflect helix transitions between the two acidic structures.
Topics: Adenine Nucleotides; Binding Sites; Hydrogen-Ion Concentration; Mathematics; Nucleic Acid Conformation; Osmolar Concentration; Poly A-U; Poly U; Polynucleotides; Sodium Chloride; Spectrophotometry, Ultraviolet; Structure-Activity Relationship
PubMed: 238672
DOI: 10.1016/0301-4622(75)80001-9 -
Proceedings of the National Academy of... Nov 1969The introduction of rII mutations into polynucleotide ligase amber mutants (gene 30) of T4D bacteriophage results in the restoration of phage growth in the nonpermissive...
The introduction of rII mutations into polynucleotide ligase amber mutants (gene 30) of T4D bacteriophage results in the restoration of phage growth in the nonpermissive host E. coli B. When cells are mixedly infected with rII(+) -ligase(-) and rII-ligase(-) phage, growth is reduced, indicating that the rII mutations are recessive to the rII(+) alleles. Infection with the rII-ligase(-) double mutants results in nearly complete restoration of phage DNA synthesis and prevents the extensive degradation of parental phage DNA observed after infection with ligase(-) single mutants. Similar results are observed when cells are infected with ligase(-) mutants and chloramphenicol is added five minutes post infection.
Topics: Centrifugation, Density Gradient; Chloramphenicol; Coliphages; DNA, Viral; Genetics, Microbial; Ligases; Mutation; Phosphorus Isotopes; Polynucleotides; Suppression, Genetic
PubMed: 5264148
DOI: 10.1073/pnas.64.3.897 -
Journal of Bacteriology May 1971The base composition of a deoxyribonucleic acid (DNA) sample affects its intrinsic rate of renaturation. In agreement with the information of Wetmur and Davidson, it was...
The base composition of a deoxyribonucleic acid (DNA) sample affects its intrinsic rate of renaturation. In agreement with the information of Wetmur and Davidson, it was established that high guanosine plus cytosine (GC) DNA renatures faster than expected from analytical measurement of its molecular weight. A calculated correction factor of 1.8% of the observed C(0)t(.5) is required for every mole per cent GC difference from 51% GC. The correction factor is now established in the range of 32 to 65% GC. Renaturation of DNA mixtures prepared from pairs of organisms has been studied. When no similarity existed between the two organisms, the observed C(0)t(.5) of the mixture was the sum of the independently determined C(0)t(.5) values. Lack of additivity was correlated with similarities in polynucleotide sequence of the reassociating DNA molecules. A quantitative relationship was formulated to relate C(0)t(.5) values of renatured DNA mixtures to per cent binding ("homology"). Finally, it was demonstrated that DNA prepared from log-phase cells renatures faster than stationary-phase DNA and also departs from theoretical second-order kinetics.
Topics: Autoradiography; Bacteria; Chromosomes, Bacterial; Cytosine; DNA Replication; DNA, Bacterial; Escherichia coli; Filtration; Genetics, Microbial; Hot Temperature; Molecular Biology; Molecular Weight; Nucleic Acid Denaturation; Nucleosides; Polynucleotides; Salmonella typhimurium; Shigella; Statistics as Topic; Vibration
PubMed: 4929869
DOI: 10.1128/jb.106.2.608-614.1971 -
European Journal of Biochemistry Nov 1992Self-deconvolution and the fourth derivative of ultraviolet absorption spectra have been used to study stacked single-stranded and double-helix structures of different...
Self-deconvolution and the fourth derivative of ultraviolet absorption spectra have been used to study stacked single-stranded and double-helix structures of different cytosine-containing polynucleotides for the first time. These compounds were studied under different solution conditions (pH and organic solvents) and at low temperatures. The red shift of the lower band (B2u band plus possibly some n-->pi* transition) of the absorption spectra in the cytosine-containing polynucleotides and the appearance of new peaks in the deconvoluted and derivative spectra in the 280-310 nm region are attributed mainly to cytosine-cytosine stacking interactions. In particular, the fourth-derivative peaks at wavelengths higher than 290 nm can be associated to coupling of electronic transitions of cytosine bases. The nature of the electronic transitions producing the absorption bands which are resolved in the aforementioned fourth-derivative peaks is discussed. It is concluded that the resolution-enhancement techniques used in this work, i.e. self-deconvolution and fourth derivative, complement each other and are useful methods to study structural changes of single-stranded and double-stranded polynucleotides allowing, at the same time, more information to be obtained about specific stacking interactions than classical absorption spectrophotometry.
Topics: Cytidine; Cytosine; Deoxycytidine; Hydrogen-Ion Concentration; Nucleic Acid Conformation; Poly C; Polynucleotides; Spectrophotometry, Ultraviolet
PubMed: 1446672
DOI: 10.1111/j.1432-1033.1992.tb17409.x