No abstract

SUMMARYSera from 58 children with juvenile rheumatoid arthritis were examined for the presence of antibodies to DNA topoisomerase II. Eight sera were reactive in immunoblotting with purified human topoisomerase II and a protein encoded by a cloned cDNA expressed in Escherichia coli which represents the carboxy-terminal domain of the human enzyme. In addition, the sera detect topoisomerase II in mitotic chromosomes and chromosome scaffolds. Five of the sera bind to the native enzyme in solution and deplete such solutions of the active enzyme. All eight sera also contain antibodies to nuclear antigens other than topoisomerase II.

ColEl DNA was isolated from Escherichia coli as a relaxation complex of supercoiled DNA and proteins. Treatment of the complex with either protein-denaturing agents (SDS, phenol etc.) or proteolytic enzymes converted the supercoiled DNA to an open-circular form (relaxation). The relaxation complex was separately labelled in vivo with [3H]Leu or ['4C]Leu, [35S]Met or (32P)phosphate and extensively purified. Complete hydrolysis of the relaxed complex with DNase I and P1 nuclease produced a 36-kDa protein which, we believe, is covalently bound to ColEl DNA. On the other hand, the relaxed complex was treated with tosylphenylalanylchloromethanetreated-trypsin and the DNA-peptide(s) produced was (were) isolated and digested with the nucleases as above. The resulting nucleotidylpeptide(s) was (were) isolated by DEAE-Sephadex chromatography. The only 5'-dCMP was released from the nucleotidylpeptide(s) by snake venom phosphodiesterase treatment. 0-Phosphoserine was found in acid hydrolysates of the DNA-peptide(s). We suggest that in the relaxation event the 36-kDa protein becomes covalently linked to ColEl DNA via a phosphodiester bond between dC and the serine residue.ColEl DNA belongs to a group of non-conjugative mobilizable plasmids which are not capable of independent transfer during bacterial conjugation but are transferred in the presence of a conjugative plasmid. Genetic studies have confirmed that the following sites and proteins are needed to ensure ColEl DNA mobilization: (a) the ColEl born sequence, which includes the nic site (oriT); (b) the ColEl mob region, where at least one ColEl-specified gene product, needed for mobilization, is encoded; (c) the tra-gene products of the donating plasmid, which recognize the oriT sequence in ColEl DNA and may specifically interact with the proteins of the ColEl relaxation complex, inducing relaxation and mobilization of the plasmid [l, 21. In vitro treatment of the relaxation complex, either with protein-denaturing agents such as SDS or with pronase, results in conversion of the DNA in the complex from a supercoiled to an open-circular configuration Abbreviations. PhMeS02F, phenylmethylsulfonyl fluoride; TosPheCH2C1, tosylphenylalanylchloromethane.Enzymes. DNase I (EC 3.1.21 .I); micrococcal nuclease (EC 3.1.31.1); nuclease P1 (EC 3.1.30.1);, snake venom phosphodiesterase I (EC 3.1.4.1); alkaline phosphatase (EC 3.1.3.1); RNase I (EC 3.1.27.5); pronase (EC 3.4.24.4); trypsin (EC 3.4.21.4). lication [8] or transcription [9]. This is supported by the fact that in the case of blocked vegetative replication of similar plasmids they cannot be transferred to the recipient cell [8]. Also, ethidium bromide intercalation with the plasmid DNA in vitro induces the complex relaxation followed by sitespecific nicking at oriT [lo].The site-specific nicking of the transferred DNA strand by the relaxation protein and the formation of an active and specific intermediate covalent complex between the ColEl DNA and the relaxation protein seem to be the key point in the initiation of pla...

No abstract