Synthese eines Strukturgens für das Peptidhormon Angiotensin II, Teil 9. Ligation der Teilsequenzen zum kompletten Doppelstrang

Blöcker, Helmut; Frank, Ronald; Köster, Hubert

doi:10.1002/jlac.197819780610

Cited by 5 publications

(2 citation statements)

References 22 publications

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“…Enzvmes. E.coli DNA polymerase I and T4 DNA ligase were isolated in this laboratory by H. MUller and R. Frank, respectively, according to standard methods (22,23). E.coli DNA ligase was prepared similar to published procedures (24,25).…”

Section: Methodsmentioning

confidence: 99%

“…Specific activity of DNA polymerase I: 8160 units per mg. One unit catalyses the incorporation of 10 ±mol of nucleotides into polyAdTd in 30 min at 37 C under assay conditions (22). One unit of DNA ligase converts 100 nmol (nucleotides) of poly(AdoTd) into exonuclease III-resistant covalently closed circles in 30 min at 30 C under assay conditions (23,26). Restriction endonuclease Taq I from Thermus aquaticus was a kind gift of Dr. H. Mayer, Stockheim.…”

Section: Methodsmentioning

confidence: 99%

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Enzymatic synthesis, ligation, and restriction of DNA containing deoxy-4-thiothymidine

Höfer

Köster

1981

Nucl Acids Res

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Phage fd RF I DNA1 about 90% substituted by deoxy-4-thiothymidine (s4Td) in the codogenic strand was synthesized by the simultaneous actions of DNA polymerase I and DNA ligase. While the rate of DNA synthesis was considerably reduced, the yield the rate of DNA synthesis was considerably reduced, the yield was not affected in the presence of s4TdTP. The conversion of RF II to RF I DNA by DNA ligase was even improved. This effect seems to be related with an altered ratio of affinity of polymerase and ligase for the s4Td-containing substrate. The presence of the base analogue in the DNA was verified independently by chromatographic and spectroscopic methods. The modified genome could be cleaved by restriction endonucleases Hpa II (C/CGG)d and Taq I (T/CGA)d. A number of the fragments produced showed altered mobilities under the conditions of polyacrylamide gel electrophoresis.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Enzymatic synthesis, ligation, and restriction of DNA containing deoxy-4-thiothymidine

Höfer

Köster

1981

Nucl Acids Res

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Synthetic Genes as a Powerful Tool for Protein Structure-Function Analysis

Frank

Blöcker

1986

Advanced Methods in Protein Microsequence Analysis

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Elimination of promoter function by base modification of DNA

Höfer¹,

Köster²

1979

FEBS Letters

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Promoters are DNA regions at which higtrty selective RNA polymerase binding and RNA chain initiation take place. They are not characterized by a unique primary structure, Compa~son of published promoter sequences [ I-141 reveals that there is not even an absolute requirement for a particular base at any positian within such a region.In view of this situation we started to examine the dependence of promoter function on specific structural elements of the nucleobases. Thus we 'removed' the 5.methyl of de~xythym~d~ne (Td) by replacement of this nucleoside by deoxyu~dine (II&l. The moditication was introduced into the codogenic strand of Escherichia coli phage fd RF DNA. Individual promoters were separated by cleavage of the circular DNA with restriction endonuclease &&rII and polyacrylamide gel electrophoresis. E. coZi RNA polymerase binding assays in the absence and presence of ribonudeoside tr~ph~spllates revealed that upon the DNA modification described the fd gene II promoter loses its affinity for the enzyme. [ 19,20] with the modi~cat~on that phages were pulled by two successive precipitations with 3% PEG in 0.5 M NaCl [2 11. fd-specific oligonucleotide primers of chain length 1 I -13 o~gin~ting from a D&se digest of fd RF DNA were prepared and purified by D. Miiller. Ribo-and deo~yribonucleoside triphosphates were from Boehringer Mannheim. [~Y-~~P] AdTP was purchased from the Radiochemic~ Centre, Amersham. RF DNA was synthesized in vitro in the presence of [o-32P]AdTP. Synthesis and isolation were done essentially as in [22]. 100% substitution of Td by Ud within the codogenic strand was achieved by replacement of TdTP by IJdTP and of DNA polymerase I by its large fragment, Detailed descriptions of these procedures will be published elsewhere (in preparations. For fra~entation of fd RF, 10 ~.rg normal RF or 5 #g modified RF were incubated with 15 units or 25 units, respectively, of NpaII at 37°C for 10 h in 0.5 ml of 30 mM Tris B HCl (pH 7.9, 10 mM MgClz, 1 mM DTE, 3 9% glycerol. The fragments were deproteinized by 2 phenol extractions and desalted on Sephadex G-50. RNA polymerase binding experiments were performed in 20 mM Tris . HQ (pH g.O), 10 mM MgQ, 120 mM RCI, 0.1 mM DTE, 0.1 mM EDTA, 5% glycerol at 37OC Mate&Is and methods DNA[23]. To allow RNA chain initiation GTP, ATP and UTP were present in some experiments at 0.1 mM

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Synthese eines Strukturgens für das Peptidhormon Angiotensin II, Teil 9. Ligation der Teilsequenzen zum kompletten Doppelstrang

Cited by 5 publications

References 22 publications

Enzymatic synthesis, ligation, and restriction of DNA containing deoxy-4-thiothymidine

Enzymatic synthesis, ligation, and restriction of DNA containing deoxy-4-thiothymidine

Synthetic Genes as a Powerful Tool for Protein Structure-Function Analysis

Elimination of promoter function by base modification of DNA

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