The development of a short incubation model of scrapie (strain 263K), in golden hamsters has added impetus to the purification of the infectious agent. Our own attempts have been based on methods pioneered by Millson and developed by Prusiner. We present here results indicating that a purification factor of up to 10(4) with respect to protein may now be possible. Fractions from brain with high infectivity had a sedimentation range of 70-300S and contained an abundance of fibrils closely similar to the scrapie-associated fibrils (SAF) discovered by Merz et al.. Material of molecular weight (Mr) 26,000, which is probably protein, appears to be a major constituent of the fibrils. The association between infectivity and fibrils raises two possibilities: the fibrils are an infectious form of the scrapie agent or they are a pathological response to scrapie infection.
Most tra proteins encoded by the Escherichia coli F sex factor are incorporated into the minicell envelope. We have now assigned the tra proteins to cytoplasm (TraIp and 2b), inner membrane (TraEp, TraMp, and TraSp), and outer membrane (6e, TraAp, TraBp, TraJp, TraKp; TraLp, and TraTp). Two proteins, TraDp and 6d, were associated with both inner and outer membranes. The proteins exported to the inner or outer membranes did not undergo proteolytic cleavage (processing) whereas P-lactamase was processed normally.Many DNA transfer (tra) cistrons encoded by the Escherichia coli F sex factor have been defined (1,2). The F DNA carrying these cistrons has been cloned to yield hybrid plasmids (3, 4). The tra proteins were detected by radioactive labeling of minicells into which the plasmid DNAs had segregated or by adding purified plasmid DNA to an in vitro protein-synthesizing system (5). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis allowed the assignment of the proteins to tra cistrons (5-10). Most of the proteins were incorporated into the envelope of the minicells (5). In contrast, only one protein, TraTp, was detectable in normal cell envelopes (6). (The other tra proteins are synthesized too slowly or degraded too quickly for detection in the presence of cell proteins.) TraTp is an outer membrane protein present in numerous copies per cell (6). It is in part responsible for converting cells that carry the F factor into poor recipients in conjugation (surface exclusion). The other tra proteins are responsible for other aspects of conjugation (1). To understand these aspects better, we decided to separate the inner and outer membranes of minicells that contained the radioactively labelled tra proteins.It already had been noted (6) that TraTp has the same apparent molecular weight whether solubilized from outer membranes or synthesized in vitro. This result suggests that TraTp is exported to the outer membrane without proteolytic processing, unlike other periplasmic and outer membrane proteins (11-16). It seemed important to test the other proteins in this regard and to exclude possible experimental artifacts. MATERIALS AND METHODSMost of the techniques used here have been described (5-7, 17).Minicells were purified from derivatives of strain DS410 (18) carrying chimeric plasmids. We have named the tra proteins according to the cistrons encoding them (e.g., TraTp for the traT gene product) or, when unknown, according to the EcoRI DNA fragment encoding them (fragment f6 for 6c, 6d, 6e, and f2 for 2b). 2b was formerly incorrectly referred to as E7-A (9).Purified f3-lactamase was a gift from R. P. Ambler and G. W.Ross.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 4837Purification of Minicells. Four 2-liter erlenmeyer flasks, each containing 500 ml of L broth (19), were inoculated with 0.1 ml from cells stored in 30% (vol/vol) glycerol/...
An Escherichia coli K12 dnaB dnaC mutant was constructed by P1 transduction of the dnaC allele into a dnaB recipient stain. dnaB dnaC transductant were discriminated from dnaB mutants by their inability to grow at 40 degree C after lysogenization with phage P1bac. The dnaB dnaC mutant character was verified by 1. P1 transduction, and 2. by in vitro complementation with dnaB and dnaC wild type protein fractions. DNA synthesis was studied in strains containing dnaB, dnaB dnaC alleles in an otherwise uniform genetic background with the dnaB character either unsuppressed or suppressed by P1bac prophage. Degradation at 42 degree C of [3H]-thymidine pulse-labeled DNA in dnaB and dnaB dnaC mutants is suppressed by P1bac. However, unlike the dnaC mutant, the P1bac lysogen of the dnaB dnaC mutant exhibits an abrupt cessation of DNA synthesis and less residual cell divisions at 42 degree C indicating an inhibition of DNA chain elongation rather than a defect in DNA initiation. It is suggested that denaturation of the dnaB protein effects the dnaC function.
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