DNA binding protein from Streptomyces hygroscopicus: detection of binding by gel retardation, sedimentation and effects on the transcriptional activity in vitro.

The binding to supercoiled plasmid DNA as well as to double-stranded short DNA fragments of the DNA binding protein. ASBIII, from S. hygroscopicus has been demonstrated by gel retardation asay. As further revealed by sedimentation analysis, the protein-DNA complex formation also involves condensation of DNA and an aggregation step at higher total protein-to-DNA ratio (w/w). In vitro studies of the transcriptional activity of DNA-protein complexes showed that the ASBIII protein inhibits the overall template activity in the RNA polymerase II system to nearly similar extents for various DNA's. Preincubation of ASBIII-DNA complexes of different GC content, however, suggested a selective inhibitory effect on GC-rich DNA due to the higher stability of this complex. The results also indicate that the initiation of transcription of the GC-rich DNA template is affected by the ASBIII protein. The present results on the DNA binding properties and inactivation of the transcription of the ASBIII protein suggest that this protein may be a potential candidate for the local compaction of chromosomal DNA in Streptomyces hygroscopicus.

Analysis of the nourseothricin-resistance gene (nat) of Streptomyces noursei.

A gene (nat) conferring resistance to the streptothricin antibiotic nourseothricin (Nc) was cloned from the producer Streptomyces noursei into Streptomyces lividans on the vector pIJ702 to form pNAT1. The nat gene was localized on a 1-kb SalI-MboI fragment, which also carries the nat promoter. Divergent promoter activity from the nat promoter region was identified on the cloned fragment using promoter probe plasmids pIJ486 and pIJ487. The nat gene is not expressed from its own promoter in Escherichia coli as shown by its failure to promote cat expression in promoter-less plasmid pBB100 and by the expression of NcR in only one orientation, when cloned in pUC19. In S. lividans 7A, harbouring plasmid pNAT1, an Nc-acetylating activity (NAT) was associated with the cloned resistance gene. The substrate specificity of NAT correlated well with the substrate range of the acetyltransferase in S. noursei and Tn1825-determined streptothricin resistance in Gram-negative bacteria. Moreover, an extract of S. lividans carrying pNAT1 showed specific serological cross-reactivity with an extract of E. coli carrying Tn1825.

Counterion dependent variation of DNA secondary structure in (A . T) clusters: evidence by use of netropsin as a structural probe.

The interaction of the oligopeptide antibiotic netropsin (Nt) with (A . T) regions of DNA is characterized by a spectrum of discrete modes. This has been revealed by viscometric analysis, at 20 degrees C and 0.2 M "counterions", for NaDNA in a preceding and for NH4DNA in this paper. The increase of DNA contour length as induced by one Nt molecule was found to depend on the special mode only, while the respective stiffening is generally higher for NH4DNA. The latter property is interpreted in terms of an enhanced flexibility, relative to that of NaDNA, of the (A . T) cluster segments before complex formation. For some of the interaction modes of the DNA-Nt systems a difference in the number of corresponding binding sites has been observed. This phenomenon is understood by assuming an influence of the counterion species upon existing equilibria between different forms of the (A . T) cluster secondary structure. Not less than 5 to 10% of the total DNA are effected in this manner. Upper limits for the local differences in the axial rise per base pair are 0.04 nm and 0.02 nm.

Properties of Thermoactinomyces vulgaris phage Ta1 and its extracted DNA.

The virulent phage Ta1 was obtained in good yields from infected cultures of Thermoactinomyces vulgaris 1227. The purified phage was found to sediment with a single band, the sedimentation constant being (519 +/- 14)S, and to exhibit a typical nucleoprotein behaviour in UV-spectrophotometric and CD experiments. The Ta1 phage consists of a hexagonal head about 0.056 micrometers in diameter and a very short tail. It is morphologically similar to the temperate Salmonella phage P22. The nucleic acid extracted from the phage was found to be a double-stranded linear DNA with a G+C content of 42 mole-% as deduced both from its melting temperature and buoyant density in CsCl. Analytical sedimentation revealed a high degree of molecular homogeneity of Ta1 Dna. the sedimentation constant of this DNA amounts to (35.9 +/- 0.3)S, corresponding to a DNA molecular weight of about 29 millions daltons. The biological activity of Ta1 DNA was indicated by its ability to infect the mycelium of the components T. vulgaris strain 1227 and to give rise to mature phages.