PMC

Transcriptional start points of the repSA gene. The sequence upstream and downstream of the repSA gene start codon is presented (positions 5761 to 6118 in the EMBL sequence). The positions of the two transcriptional start points are indicated by +1, followed by the number corresponding to the signal numbers on Fig. and also marked by vertical arrows. The positions of the presumed start and stop translation codons and the restriction sites EcoRI and NotI are indicated. The numbers in parentheses correspond to their positions in Fig. . RBS, ribosome binding site; oligo, oligonucleotide.

Map of pOS548 showing the pSAM2 genes and open reading frames and the attP and ori⁺ sites. The pra gene promoter was replaced by the fragment containing the phage fd transcriptional terminator (term fd) and the tipA promoter (ptipA) inducible by nosiheptide. The two resistance genes, tsr (conferring resistance to nosiheptide []) and hyg (conferring resistance to hygromycin []) are shown. The pBR322 replicon allows the maintenance of pOS548 in E. coli and carries an ampicillin resistance gene. The number in parentheses refers to the nucleotide position of the site.

High-resolution S1 mapping of the repSA gene transcriptional start. To obtain a ssDNA corresponding to the presumed promoter region of the repSA gene, the 1.16-kb BclI(18533)-EcoRI(19700) (Fig. ) fragment of pSAM2 was cloned in the BamHI and EcoRI sites of the M13mp18 vector and its ssDNA was isolated. To synthesize the second strand, this ssDNA was annealed with labelled oligonucleotide AMD3. Synthesized double-stranded DNA was directly digested with EcoRI, giving a labelled fragment of 537 bp. Total RNA was hybridized with this DNA fragment and treated with S1 nuclease. Lane 1, DNA probe treated with S1 enzyme in the presence of total RNA from S. lividans/pSAM2_B3; lane 2, DNA probe treated with S1 enzyme in the presence of total RNA from S. lividans/pOS11Δ; lane 3, DNA probe treated with S1 enzyme in the absence of total RNA. The sizes of the revealed protected DNA fragments were determined with the sequence of the BclI(18533)-EcoRI(19700) fragment (Fig. ; fragment 6641 to 5477 in the EMBL sequence) cloned in M13mp18 and sequenced with the standard −40 oligonucleotide.

Effect of pra gene expression on the status of pOS546 in S. lividans. (A) Appearance of the free form of pOS546. Total DNA digested by EcoRI was analyzed by Southern hybridization with the ³²P-labelled EcoRI(15493)-EcoRI(19700) pSAM2 fragment (Fig. ). The total DNA was extracted from S. lividans (lanes 1 to 4) and from S. lividans/pOS689 (lanes 5 and 6) containing pOS546 and grown in the presence or absence of inducer. The 6.7- and 5.2-kb fragments correspond to pOS546 integrated at the attB site. The 4.2-kb fragment corresponds to the free form of pOS546. Lane 1, 0.1 μg of nosiheptide ml⁻¹, clone 1; lane 2, 0.1 μg of nosiheptide ml⁻¹, clone 2; lane 3, no nosiheptide, clone 1; lane 4, no nosiheptide; lane 5, no nosiheptide, clone 1; lane 6, 0.1 μg of nosiheptide ml⁻¹, clone 1. (B) Appearance of the unoccupied attB site. Total DNA from S. lividans/pOS546 digested by PstI was analyzed by Southern hybridization with the ³²P-labelled 40-mer oligonucleotide probe OL-1. Total DNA was extracted from S. lividans/pOS546 grown as follows. Lane 1, with inducer, clone 1; lane 2, with inducer, clone 2; lane 3, no inducer, clone 1; lane 4, no inducer, clone 2; lane 5, S. lividans TK24 (no plasmid). For details, see the legend to Fig. .

Effect of pra gene expression on the status of pOS548 in S. lividans and S. lividans/pOS689. (A) Appearance of the free form of pOS548. Total DNA digested by EcoRI was analyzed by Southern hybridization with the ³²P-labelled EcoRI(15493)-EcoRI(19700) pSAM2 fragment (Fig. ). Total DNA was extracted from S. lividans (lanes 1 to 4) and from S. lividans/pOS689 (lanes 5 to 8) containing pOS548 and grown in the presence or absence of nosiheptide as tipAp inducer. The 6.7- and 5.2-kb fragments indicate the presence of pOS548 integrated at the attB site. The 4.2-kb fragment indicates the presence of the free form of pOS548. Lane 1, no nosiheptide; lane 2, 0.01 μg of nosiheptide ml⁻¹; lane 3, 0.1 μg of nosiheptide ml⁻¹; lane 4, 1.0 μg of nosiheptide ml⁻¹; lane 5, no nosiheptide, clone 1; lane 6, no nosiheptide, clone 2; lane 7, 0.1 μg of nosiheptide ml⁻¹, clone 1; lane 8, 0.1 μg of nosiheptide ml⁻¹, clone 2. (B) Appearance of unoccupied attB sites. Total DNA from S. lividans/pOS548 digested by PstI was analyzed by Southern hybridization with the ³²P-labelled 40-mer oligonucleotide probe OL-1 that corresponds to a part of the identity segment between the S. lividans attB and the pSAM2 (and pOS548) attP sites (). Total DNA was extracted from S. lividans/pOS548 grown in the presence (lane 1) or absence (lane 2) of inducer. The positions of attB, attR, and attL are indicated by arrows. The unoccupied attB site is situated in a 7.5-kb chromosomal PstI DNA fragment. If the attB site was occupied by pOS548, fragments of 6.3 and 21.0 kb containing the attL and attR sites, respectively, were detected. attP and attR are carried by fragments of 19.75 and 21.0 kb, respectively, that were not resolved in the gel. The position of ssDNA is also indicated.