Transcriptional levels of the Escherichia coli mioC and gidA genes, which flank the chromosomal origin of replication (oriC) and the dnaA gene, were correlated with the time of initiation of chromosome replication. The transcripts were measured either in dnaC2(ts) mutants that had been aligned for initiation of chromosome replication by a temperature shift or in synchronous cultures of cells obtained using the baby machine technique. In both types of experiments, mioC transcription was inhibited prior to initiation of chromosome replication and resumed several minutes after initiation. Conversely, gidA and dnaA transcription were both inhibited after initiation of replication, coincident with the period of hemimethylation of oriC DNA. It is proposed that mioC transcription prevents initiation of chromosome replication, and must terminate before replication can begin. It is further proposed that the eclipse period between rounds of replication, i.e. the minimum interval between successive initiations, encompasses the time required to methylate GATC sequences in newly replicated oriC plus the time required to terminate mioC transcription. Conversely, the active transcription of gidA and dnaA prior to initiation is consistent with their positive effects on initiation, and their shutdown after initiation could serve to limit premature reinitiation.

The infectivity of hamster scrapie strain 263K was measured in platelets isolated from blood pooled from six hamsters with clinical scrapie. The total number of infectious doses present in the blood pool was 220, out of which only 3.5 infectious doses were associated with platelets. A larger proportion of the total infectivity was recovered from the mononuclear leukocyte fraction. This result indicates that platelets are not the source of blood-borne infectivity in transmissible spongiform encephalopathy-infected hamsters.Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases of both humans and animals (5). TSE infectivity is present in the blood of experimentally infected rodents showing symptoms of the disease, albeit at very low levels (3,4,9). This infectivity partitions to both cellular and plasma fractions during component separation in a manner suggesting an association with blood platelets, which frequently contaminate other blood fractions (3, 4; Rohwer laboratory, ongoing studies). Moreover, human platelets contain a significant concentration of the cellular prion protein, PrP c (7, 10). Much lower concentrations are present in rodent platelets (8).To measure the concentration of TSE infectivity in platelets, we purified platelets from the pooled blood of six hamsters in the terminal stages of scrapie after intracerebral inoculation with a dilution of hamster scrapie strain 263K brain homogenate containing approximately 10 2 infectious doses (ID) per inoculum. Low-titer inoculation was utilized to prevent any chance of reisolating the inoculum itself in the blood collected 144 days later during clinical disease. The hamsters were anesthetized, their chest cavities were opened, and blood was collected into 3.8% sodium citrate (9:1) by cardiac puncture, with great care being taken not to expose the needle to any other tissue. A total of 22 ml of blood in 2-ml aliquots was collected. Platelets were prepared by a method previously optimized for hamster blood (8). The blood of individual hamsters was diluted with an equal volume of physiological saline (PS) and centrifuged through Ficoll. The cellular band containing platelets and mononuclear leukocytes was collected from the top of each gradient, and the pooled fraction was diluted with 30 ml of washing buffer (12.9 mM sodium citrate, 30 mM glucose, 120 mM NaCl, 5 mM EDTA [pH 6.5]). The mononuclear cells were pelleted from the suspension by centrifugation at 250 ϫ g for 5 min. The supernatant was collected, and residual mononuclear cells were removed by a second centrifugation at 250 ϫ g for 5 min. The platelets in the supernatant from the second spin were then pelleted by centrifugation at 1,200 ϫ g for 15 min. The platelet pellet was washed twice to remove residual plasma and Ficoll. The concentration of Ficoll or plasma in the final platelet fraction was less than 0.01%. The washed platelets were resuspended in 1 ml of PS and counted with a hemocytometer. The final preparation contained 5.2 ϫ 10 9 platelets in 1 ml of PS, ...

Exponentially growing derivatives of Escherichia coli K-12 were immobilized onto the surfaces of nitrocellulose membrane filters which had been coated with poly-D-lysine. The cells attached firmly to the surfaces, and when flushed with culture medium, the immobilized cells continued to divide and newborn cells were released into the effluent. Cell cycle parameters were examined with the technique, and it was found that K-12 derivatives possessed differing values for interdivision times, C, D, and average cell sizes when grown in the same culture media. It was also found that the cells released from immobilized populations of one culture consisted of two predominant size classes: newborn cells of unit size with single nucleoids and newborn cells of double this unit size. The results demonstrated that K-12 derivatives can be used in the baby machine culture technique to examine all aspects of the cell cycle of this organism. Furthermore, the yield of newborn cells was about fivefold greater than that obtained previously with cultures of strain B/r immobilized onto uncoated membranes.