The rate of liberation of free acetylcholine from the surface of prostigmin-treated cerebral cortex in the freely moving cat has been determined in states of slow wave sleep, paradoxical or activated sleep, and waking. The average rate during slow wave sleep (1.2 nanograms per minute per square centimeter of cortical surface) increased during paradoxical sleep (2.2 nanograms per minute) and during waking (2.1 nanograms per minute). The rate of acetylcholine release is thus related to the electroencephalogram pattern of desynchronized activatin of the cortex rahter than to the behavioral responsiveness of the animals.

Minisatellites are tandemly repeated DNA sequences of 10-100-bp units. Some minisatellite loci are highly unstable in the human germ line, and structural analysis of mutant alleles has suggested that repeat instability results from a recombination-based process. To provide insights into the molecular mechanism of human minisatellite instability, we developed Saccharomyces cerevisiae strains carrying alleles of the most unstable human minisatellite locus, CEB1 (ref. 2). We observed that CEB1 is destabilized in meiosis, resulting in a variety of intra- and inter-allelic gains or losses of repeat units, similar to rearrangements described in humans. Using mutations affecting the initiation of recombination (spo11) or mismatch repair (msh2 pms1 ), we demonstrate that meiotic destabilization depends on the initiation of homologous recombination at nearby DNA double-strand break (DSBs) sites and involves a 'rearranged heteroduplex' intermediate. Most of the human and yeast data can be explained and unified in the context of DSB repair models.

This study identifies a region of the adeno-associated virus type 2 (AAV-2) rep gene (nucleotides 190 to 540 of wild-type AAV-2) as a cis-acting Rep-dependent element able to promote the replication of transiently transfected plasmids. This viral element is also shown to be involved in the amplification of integrated sequences in the presence of adenovirus and Rep proteins.It was previously reported that efficient recombinant adenoassociated virus (AAV) production using stable rep-cap cell lines correlated with a 100-fold amplification of the AAV-2 genes upon adenovirus infection (3,9). This phenomenon, which occurred despite the absence of inverted terminal repeats (ITRs) generated extrachromosomal double-stranded DNA molecules harboring the rep-cap genes and required the activity of the adenovirus DNA binding protein, cellular polymerases, and Rep proteins (9). A question that remained unanswered was whether the rep-cap amplification was dependent on the activity of an as-yet-unidentified viral origin of replication present within the viral genome.To answer this question, we investigated if a rep-cap-containing plasmid was able to replicate following transient transfection into adenovirus-infected cells. 293 cells were transfected with plasmid pRCtag containing the rep-cap genome with the ITRs deleted, ligated to a 3Ј tag sequence, and then mock or adenovirally infected. After DNA extraction, replication was assessed by digestion with DpnI or MboI followed by Southern blot analysis using a tag probe (Fig. 1). Cleavage by DpnI indicates that both strands are methylated in the absence of replication of the transfected DNA; cleavage by MboI occurs only if both strands are unmethylated as a result of two rounds of replication. In the absence of adenovirus, the pRCtag plasmid did not replicate (Fig. 1B, lanes 5 and 6). In contrast, upon adenoviral infection, a fraction of the plasmid DNA was susceptible to MboI digestion (Fig. 1B, lane 9), indicating that some input rep-cap molecules had replicated. After DpnI digestion, high-molecular-weight resistant bands were detected as weak signals, suggesting that pRCtag replication generated products which are heterogenous in size (Fig. 1B, lane 8).To identify the cis element(s) involved in pRCtag replica- , and analyzed on a Southern blot by using a tag probe. As a control (lanes 1, 2, and 3), untransfected pRCtag plasmid DNA mixed with 10 g of total DNA from 293 cells was digested with DpnI or MboI and similarly analyzed using the tag probe. The expected 1,430-bp DpnI-MboI fragment hybridizing to the tag probe is indicated. 9991on May 12, 2018 by guest

We previously reported that a 350-bp region of the adeno-associated virus (AAV) type 2 rep gene contains a cis-acting element responsible for the Rep-dependent replication of a transiently transfected rep-cap plasmid. In this study, we further report that replicated rep-cap sequences can be packaged into AAV capsids in the absence of the inverted terminal repeats.The usual procedure for recombinant adeno-associated virus (rAAV) assembly involves transfection of the vector and the rep-cap plasmid into cells which are either infected with adenovirus or cotransfected with an adenoviral helper plasmid (4,11,15). Despite the lack of homologous sequences between the rep-cap and vector sequences, the precise characterization of rAAV preparations indicated that they were contaminated to various extents with particles containing rep-cap AAV sequences. We designated these contaminating particles rep positive (rep ϩ ), because they were able to transfer a Rep function, as detected by a replication center assay (RCA) (10). Previous studies have indicated that most of these particles were replication competent and that they arose from nonhomologous recombination events between the rep-cap plasmid and the inverted terminal repeats (ITRs) in the rAAV vector (1, 14). Deletion of critical ITR sequences involved in the nonhomologous recombination events prevented the formation of such replication-competent particles. However, rAAV preparations remained contaminated by replication-defective AAV particles containing rep-cap genomes, further suggesting that these sequences had been packaged in the absence of the ITRs (14).Altogether, these observations suggested that some additional cis-acting elements were present in the rep-cap sequences, allowing their replication and encapsidation. We and others have previously reported the presence of a cis-acting replication element (CARE) located in the 5Ј portion of the rep gene (9, 12). The CARE was localized in a 350-bp region that included the p5 promoter and the 5Ј portion of the rep coding sequence (nucleotides 190 to 540 of wild-type AAV), and it was demonstrated that this element behaved in vitro and in vivo as a Rep-dependent origin of replication in the absence of both ITRs (9).In the present study, we investigated whether the presence of CARE could also lead to the packaging of rep-cap sequences into AAV capsids in the absence of the viral ITRs. A critical element in this study was the need to distinguish between DNA truly packaged inside the particles and that simply contaminating the preparations. The conventional procedure to extract viral DNA from a cell lysate or a purified rAAV stock relies on the use of DNase I to digest contaminating DNA before extraction of packaged sequences. The activity of DNase I was checked by mixing purified rAAVLZ particles (rAAV encoding the nucleus-localized ␤-galactosidase) with up to 400 ng of X174 DNA (either double or single stranded). After digestion for 1 h at 37°C with 50 U of DNase I (Roche) in 500 l of Dulbecco modified Eagle medium, pack...