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Journal ArticleDOI

Studies on Polynucleotides, C A Novel Joining Reaction Catalyzed by the T4-Polynucleotide Ligase

01 Nov 1970-Proceedings of the National Academy of Sciences of the United States of America (National Academy of Sciences)-Vol. 67, Iss: 3, pp 1468-1475
TL;DR: The polynucleotide ligase isolated from T4-infected Escherichia coli is shown to bring about repair of breaks in the single strands of bihelical DNA and can also catalyze the joining of DNA duplexes at their base-paired ends.
Abstract: The polynucleotide ligase isolated from T4-infected Escherichia coli was previously shown to bring about repair of breaks in the single strands of bihelical DNA. The present work shows that the enzyme can also catalyze the joining of DNA duplexes at their base-paired ends. This novel reaction occurs when the deoxynucleoside at a 5′-end carries a phosphate group and the complementary deoxynucleoside opposite to it carries a 3′-hydroxyl group. The consequence is the lengthening of the original duplex to form dimers or oligomers depending upon whether one or both ends are base-paired.
Citations
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Journal ArticleDOI
17 Jun 1977-Science
TL;DR: Recombinant bacterial plasmids have been constructed that contain complementary DNA prepared from rat islets of Langerhans messenger RNA that contain cloned sequences representing the complete coding region of rat proinsulin I, part of the preproinsulin II prepeptide, and the untranslated 3' terminal region of the mRNA.
Abstract: Recombinant bacterial plasmids have been constructed that contain complementary DNA prepared from rat islets of Langerhans messenger RNA. Three plasmids contain cloned sequences representing the complete coding region of rat proinsulin I, part of the preproinsulin I prepeptide, and the untranslated 3' terminal region of the mRNA. A fourth plasmid contains sequences derived from the A chain region of rat preproinsulin II.

1,135 citations

Journal ArticleDOI
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

Journal ArticleDOI
I. R. Lehman1
29 Nov 1974-Science
TL;DR: A steady state kinetic analysis of the reaction-catalyzed E. coli ligase supports this mechanism, and demonstrates that enzyme-adenylate and DNA-adenyate are kinetically significant intermediates on the direct path of phosphodiester bond synthesis.
Abstract: DNA ligase of E. coli is a polypeptide of molecular weight 75,000. The comparable T4-induced enzyme is somewhat smaller (63,000 to 68,000). Both enzymes catalyze the synthesis of phosphodiester bonds between adjacent 5'-phosphoryl and 3'-hydroxyl groups in nicked duplex DNA, coupled to the cleavage of the pyrophosphate bond of DPN (E. coli) or ATP (T4). Phosphodiester bond synthesis catalyzed by both enzymes occurs in a series of these discrete steps and involves the participation of two covalent intermediates (Fig. 1). A steady state kinetic analysis of the reaction-catalyzed E. coli ligase supports this mechanism, and further demonstrates that enzyme-adenylate and DNA-adenylate are kinetically significant intermediates on the direct path of phosphodiester bond synthesis. A strain of E. coli with a mutation in the structural gene for DNA ligase which results in the synthesis of an abnormally thermolabile enzyme is inviable at 42 degrees C. Although able to grow at 30 degrees C, the mutant is still defective at this temperature in its ability to repair damage to its DNA caused by ultraviolet irradiation and by alkylating agents. At 42 degrees C, all the newly replicated DNA is in the form of short 10S "Okazaki fragments," an indication that the reason for the mutant's failure to survive under these conditions is its inability to sustain the ligation step that is essential for the discontinuous synthesis of the E. coli chromosome. DNA ligase is therefore an essential enzyme required for normal DNA replication and repair in E. coli. Purified DNA ligases have proved to be useful reagents in the construction in vitro of recombinant DNA molecules.

607 citations

Journal ArticleDOI
08 Dec 1977-Nature
TL;DR: The primary structure of DNA containing the sequence for rat pituitary growth hormone mRNA has been determined and the amino acid sequences for rat growth hormone and its precursor form have been deduced from the determined nucleotide sequences.
Abstract: The primary structure of DNA containing the sequence for rat pituitary growth hormone mRNA has been determined. DNA was obtained by reverse transcription of polyadenylated RNA from cultured pituitary cells and from recombinant bacterial plasmids. The amino acid sequences for rat growth hormone and its precursor form have been deduced from the determined nucleotide sequences.

509 citations

Journal ArticleDOI
TL;DR: By techniques of recombination in vitro, a plasmid bearing the repressor gene (cI) of bacteriophage lambda fused to the promoter of the lac operon is constructed.
Abstract: By techniques of recombination in vitro, we have constructed a plasmid bearing the repressor gene (cI) of bacteriophage lambda fused to the promoter of the lac operon. Strains carrying this plasmid overproduce lambda repressor. This functional cI gene was reconstituted by joining DNA fragments bearing different parts of that gene. Flush end fusion techniques, involving no sequence overlap, were necessary for the construction; in certain cases, the abutting of the DNA molecules bearing ends generated by different restriction endonucleases creates a sequence at the junction which is recognized by one of the restriction endonucleases.

346 citations