Chemische Synthese der Oligonucleotide dpG‐A‐T‐A‐T‐C, dpG‐A‐T‐C‐T‐T‐T‐T und dpT‐T‐T‐C‐A‐T‐C‐A‐T

Phage fX174 A* protein cleaves singlestranded DNA and then binds to the 5'-phosphorylated terminus of the cleaved DNA fragment, forming a covalent protein-DNA complex. The bound A* protein can religate the termini to form covalently closed single-stranded circles. To determine the nature of the covalent linkage and the amino acid involved, we used A* protein to cleave DNA synthesized in vitro with [a-32P]dATP to form the A*-single-stranded DNA complex. The complex was then digested with DNase I and the 32P-labeled A* protein was isolated by electrophoresis on polyacrylamide gels. The isolated complex was digested with either trypsin or Pronase. Incubation of the tryptic digest with snake venom phosphodiesterase gave 32P-labeled products that migrated on electrophoresis on cellulose plates to the cathode, indicating covalent linkage of 32P-labeled dAMP residues to a tryptic peptide. (18)(19)(20). The isolation of an active A*-ss DNA complex in which the bound protein can religate the 5' and 3' termini to produce covalently closed singlestranded circles (19) and the fact that the 5' ends of the complex were blocked to the action of bacterial alkaline phosphatase and polynucleotide kinase (20) suggest that the A* protein binds covalently to the 5'-phosphorylated ends of the cleaved DNA. In the experiments described here, we elucidated the covalent linkage between the A* protein and the 5'-phosphorylated terminus of the cleaved ss DNA fragment. MATERIALS AND METHODSEnzymes. Snake venom phosphodiesterase, DNase I, and bacterial alkaline phosphatase were from Worthington. Bacterial alkaline phosphatase was further purified as described (21). The A* protein was purified from 4X174-infected E. coli cells as a by-product of the purification of OX gene A protein (10). Steps 1-5 were as described (10). The fraction that did not bind to hydroxyapatite contained the A* protein. This fraction was further purified through a heparine-Sepharose column. The protein was assayed by its ability to cleave ss DNA (18,19), the capacity to inhibit the OX stage II reaction in vitro (17), and by its mobility as a 34-to 36-kDa protein on polyacrylamide gels. Details of the purification scheme will be published elsewhere.Synthesis of [a-32P] 4285The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Section: Methodsmentioning

confidence: 99%

Cleavage of single-stranded DNA by the ϕX174 A ^* protein: The A ^* -single-stranded DNA covalent linkage

Sanhueza

Eisenberg²

1984

“…T7 DNA polymerase (80% pure) was prepared from T73,4,6-infected E. coli D10 as described by Adler and Modrich (15). Bacteriophage T4 polynucleotide kinase and E. coli alkaline phosphatase have been described (16). Restriction enzymes were obtained from New England BioLabs.…”

Section: Methodsmentioning

confidence: 99%

Template recognition sequence for RNA primer synthesis by gene 4 protein of bacteriophage T7.

Tabor¹,

Richardson²

1981

149

“…It was then digested with BamHI, and treated with Escherichia coil alkaline phosphatase (Worthington) to prevent intramolecular rejoining (5). [The commercial alkaline phosphatase had been further purified according to Weiss et al (11).] Plasmid and double-stranded DNAs were then ligated at 16°C for 1 hr in a reaction mixture containing 60 mM Tris-HCl at pH 8.0, 8 mM MgCl2, 1 mM ATP, 10 mM dithiothreitol, cDNA at 12.5 ,g/ml, pBR322 at 125 Mg/ml, and 110 units of T4 ligase per ml.…”

Section: Methodsmentioning

confidence: 99%

Cloning of sea urchin actin gene sequences for use in studying the regulation of actin gene transcription

Merlino

Water

Chamberlain

et al. 1980

In order to investigate the regulation of actin gene transcription during early sea urchin development, a specific hybridization probe for actin sequences is required. Such a probe was produced by cloning cDNA transcribed from a sea urchin poly(A)containing mRNA preparation enriched for actin message. Double-stranded DNA was ligated into the BamHI restriction site of plasmid pBR322, and the resulting hybrid molecules were used to transform the Escherichia coli strain ML100. After preliminary screening of bacterial colonies by antibiotic sensitivity and hybridization back to the original cDNA, clones containing sea urchin DNA were further characterized by a positive translation assay in which total sea urchin mRNA was hybridized to plasmid, and the hybridized message then was eluted and translated in a reticulocyte cellfree protein-synthesizing system. In this way, one clone (pSA38) was found to hybridize selectively to sea urchin mRNA coding for a protein of 43,000 daltons. This protein was identified as actin by three criteria: electrophoretic migration in two-dimensional polyacrylamide gels, affinity for DNase I, and peptide mapping. Restriction endonuclease and heteroduplex mapping of pSA38 indicate that it contains a 1.5-kilobase-pair insert and is therefore likely to contain a large portion of the actin coding sequence. By using pSA38 as a hybridization probe, it has been found that the level of actin-specific RNA sequences increases dramatically during early sea urchin development. It has become clear in recent years that actin is a major constituent of nonmuscle cells, in which it plays an important role in governing cell shape and motility (1-3). This is especially true in the developing embryo, where differentiating cell types are continually reorienting themselves during the process of morphogenesis. Therefore, an important problem in developmental biology concerns the role of actin in early embryogenesis, particularly gastrulation.A dramatic change in the rate of actin synthesis has been observed during early sea urchin development, when it increases from an almost undetectable level at morula to one of the most prominently synthesized proteins by blastula (4). A concomitant increase in translatable actin mRNA is observed over the same time period (4). In order to assess whether this large increase in actin mRNA is due to an alteration in actin gene transcription, a hybridization probe containing actin gene sequences is required. Such a probe can be constructed by taking advantage of recent advances in recombinant DNA technology, which allow one to generate a purified probe without the requirement for a purified mRNA as starting material (5, 6). The present paper reports the successful cloning and identification of actin gene sequences and the use of this cloned material as a hybridization probe to demonstrate a marked increase in actin RNA sequences during early sea urchin development. MATERIALS AND METHODSIsolation of Sea Urchin mRNA. Polysomes prepared from blastulae of sea urchins (Strongy...