In vivo transcription of two promoters, PTH4 and PTH270 involved in regulation ofStreptomyces differentiation

The promoters, PTH4 and P-TH270 involved in the regulation of Streptomyces coelicolor differentiation were subcloned into Streptomyces promoter, i.e. probe plasmid pIJ4083, and the recombinant plasmids, pIJ4470 and pIJ4471, were constructed. Two promoters could drive the expression of reporter gene encoding catechol dioxygenase when pIJ4470 and pIJ4471 were introduced into some white mutants (C85, C70, C71, C17 and C119). The total RNA was isolated from these strains containing recombinant plasmid. Probes were prepared by labelling 5 -ends of PTH4 AND PTH270 DNA fragments using radioisotope. DNA - RNA hybridization was carried out with the probes and RNAs isolated from different strains. The S1 mapping result showed that all RNAs from strains of C85/pIJ4470, C85/4471, C70/pIJ4470, C70/pIJ4471 and C17/pIJ4470 as well as C17/pIJ4471 gave rise to strong positive hy-bridization signal, whereas RNAs from C71/pIJ4470 and C71/pIJ4471 did not give any positive signal. RNAs from C119/pIJ4470 and C119/pIJ4471 gav     

Structure and function ofsawB, a gene involved in differentiation ofStreptomyces ansochromogenes

A partial DNA library of Streptomyces ansochromogenes 7100 was constructed by using plasmid plJ702 as vector and white mutant W19 as recipient. About 3 000 clones were obtained, two of which gave rise to the grey phenotype as wild type 7100. The plasmids were isolated from two transformants. The result indicated that the 5.2 kb and 5.8 kb DNA fragments were inserted into plJ702. The resulting recombinant plasmids were designated as pNL-1 and pNL-2 respectively. The 1.25 kb Pstl l-Apa l DNA fragment from pNL-1 was recognized as its complementarity to W19 strain. The nucleotide sequence of the 3.0 kb Pst I DNA fragment including 1.25 kb was determined and analyzed. The result indicated that this DNA fragment contains one complete open reading frame (ORF1) which encodes a protein with 295 amino acid residues, and this gene was designated as sawB. The deduced protein has 81% amino acid identities in comparison with that encoded by whiH in Streptomyces coelicolor. The function of sawB gene was studied by usi     

Independent recruitment in vivo by Gal4 of two complexes required for transcription

We use a modified form of ChIP to analyze the recruitment of seven sets of proteins to the yeast GAL genes upon induction. We resolve three stages of recruitment: first SAGA, then Mediator, and finally Pol II along with four other proteins (including TBP) bind the promoter. In a strain lacking SAGA, Mediator is recruited with a time course indistinguishable from that observed in wild-type cells. Our results are consistent with the notion that a single species of activator, Gal4, separately contacts, and thereby directly recruits, SAGA and Mediator.