Phenotypes conferred by the Bacillus subtilis recM13 mutation and the din23 fusion.

The din23 fusion encodes a B. subtilis SOS-inducible regulatory region fused to the E. coli lacZ gene (Love et al., 1985). A strain encoding the din23 fusion and a recM13 allele showed low-level constitutive beta-galactosidase expression, was induced for beta-galactosidase production by DNA gyrase inhibitors but not by DNA-damaging agents, and was slightly induced by a variety of agents which do not normally induce the SOS regulon. The din23 fusion itself resulted in high levels of spontaneous prophage induction in wild-type, recM- and recA-hosts, despite the fact that the din23recM13 strain was not induced for beta-galactosidase production by DNA-damaging agents. The results suggest that the recM gene may be involved with the regulation of the RecA protease-mediated SOS response, while the din23 gene may be involved with the regulation of an alternative function which results in the cleavage of prophage repressor.

Vampire bat salivary plasminogen activator is quiescent in human plasma in the absence of fibrin unlike human tissue plasminogen activator.

The vampire bat salivary plasminogen activator (Bat-PA) is a potent PA that exhibits remarkable selectivity toward fibrin-bound plasminogen (Gardell et al, J Biol Chem 256: 3568, 1989). Herein, we describe the activity of recombinant DNA-derived Bat-PA (rBat-PA) in a human plasma milieu. rBat-PA and recombinant human single-chain tissue plasminogen activator (rt-PA) are similarly efficacious at lysing plasma clots. In stark contrast to rt-PA, the addition of 250 nmol/L rBat-PA to plasma in the absence of a clot failed to deplete plasminogen, alpha 2-antiplasmin and fibrinogen. The lytic activities exhibited by finger-domain minus Bat-PA (F- rBat-PA) and finger and epidermal growth factor-like domains minus Bat-PA (FG- rBat-PA) were less than rBat-PA, especially at low concentrations of PA; nevertheless, these truncated forms also possessed a strict requirement for a fibrin cofactor. The loss of PA activity following the addition of rBat-PA to plasma was slower than that observed when either rt-PA or two-chain rt-PA was added. The efficacy, fibrin selectivity, and decreased susceptibility to inactivation exhibited by rBat-PA in vitro in a human plasma milieu suggests that rBat-PA may be superior to rt-PA for the treatment of thrombotic complications.

The uses of computer-aided signal peptide selection and polymerase chain reaction in gene construction and expression of secreted proteins.

The computer program, SIGSEQ2, was used to select heterologous signal peptides from a catalog of published sequences to express the echistatin gene in insect (Sf9) cells. S-values for each amino acid were determined to select empirically the site of cleavage between the signal peptide and mature echistatin. Five gene fragments encoding the signal peptides for human immunoglobulin kappa (Ig kappa), Drosophila 68C glue, antistasin, bovine growth hormone (bGH), and human apolipoprotein E (Apo E) were constructed by the use of long synthetic oligonucleotides or polymerase chain reaction (PCR). Echistatin expression vectors then were constructed using the baculovirus polyhedrin promoter. Following transient transfection, the media were assayed for echistatin activity. The results indicate that the computer program greatly facilitated the selection and design of five different signal peptides and accurately predicted their relative functionality in the expression and secretion of echistatin in insect cell cultures.

Drug-induced relaxation of supercoiled plasmid DNA in Bacillus subtilis and induction of the SOS response.

Whereas treatment with many different drugs led to induction of the SOS response in Bacillus subtilis, only inhibitors of DNA gyrase subunit B and, unexpectedly, polyether antibiotics (membrane ionophores) led to relaxation of supercoiled plasmid DNA. However, treatment with DNA gyrase subunit B inhibitors but not with polyethers led to SOS induction. Thus, the presence of underwound supercoiled DNA was not sufficient to induce the SOS response. Possible mechanisms by which polyethers induce relaxation of supercoiled DNA in vivo are discussed.