No abstract

Mammalian DNA replication origins localize to sites that range from base pairs to tens of kilobases. A regular distribution of initiations in individual cell cycles suggests that only a limited number of these numerous potential start sites are converted into activated origins. Origin interference can silence redundant origins; however, it is currently unknown whether interference participates in spacing functional human initiation events. By using a novel hybridization strategy, genomic Morse code, on single combed DNA molecules from primary keratinocytes, we report the initiation sites present on 1.5 Mb of human chromosome 14q11.2. We confirm that initiation zones are widespread in human cells, map to intergenic regions, and contain sequence motifs found at other mammalian initiation zones. Origins used per cell cycle are less abundant than the potential sites of initiation, and their limited use increases the spacing between initiation events. Between-zone interference decreases in proportion to the distance from the active origin, whereas within-zone interference is 100% efficient. These results identify a hierarchical organization of origin activity in human cells. Functional origins govern the probability that nearby origins will fire in the context of multiple potential start sites of DNA replication, and this is mediated by origin interference. INTRODUCTIONEukaryotic cells have a limited amount of time, defined by the length of S phase, to duplicate their genomes. This is achieved by synthesizing DNA at replication forks, which extend from multiple sites of initiation. Because fork speed is not scaled according to S-phase length, regulating the frequency of initiation along each respective chromosome is required to prevent unreplicated gaps before the onset of mitosis (Hand and Tamm, 1973;Edenberg and Huberman, 1975).Although there are exceptions, the common view is that somatic mammalian origins fire at 50-to 300-kilobase (kb) intervals (Edenberg and Huberman, 1975;Berezney et al., 2000). This suggests that Metazoa do possess a mechanism to evenly distribute initiation events. Placing strong replicator sequences at regular distances is one such mechanism that is used by the budding yeast, Saccharomyces cerevisiae (Newlon et al., 1991;Shirahige et al., 1993). In higher eukaryotes, genetic elements play a role in origin activation; however, they are not sufficient by themselves to drive initiation (Gilbert, 2004). Furthermore, although some Metazoan origins localize to well-circumscribed sites of a few base pairs, a large number localize to more disperse initiation zones ranging up to tens of kbs (DePamphilis, 1999).This raises the problem of how to achieve a regular distribution of activated origins from a range of potential sites that possess low intrinsic efficiency.One method to regulate origin activity is to change the probability that it will be replicated passively. As an elongating fork from an origin neighbor mediates this suppression, this form of origin deactivation has been termed "origin inte...

A novel method, single-molecule anisotropy imaging, has been employed to simultaneously study lateral and rotational diffusion of fluorescence-labeled lipids on supported phospholipid membranes. In a fluid membrane composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, in which the rotational diffusion time is on the order of the excited-state lifetime of the fluorophore rhodamine, a rotational diffusion constant, D(rot) = 7 x 10(7) rad(2)/s, was determined. The lateral diffusion constant, measured by direct analysis of single-molecule trajectories, was D(lat) = 3.5 x 10(-8) cm(2)/s. As predicted from the free-volume model for diffusion, the results exhibit a significantly enhanced mobility on the nanosecond time scale. For membranes of DPPC lipids in the L(beta) gel phase, the slow rotational mobility permitted the direct observation of the rotation of individual molecules characterized by D(rot) = 1.2 rad(2)/s. The latter data were evaluated by a mean square angular displacement analysis. The technique developed here should prove itself profitable for imaging of conformational motions of individual proteins on the time scale of milliseconds to seconds.

We developed a microarray platform for PCR amplification-independent expression profiling of minute samples. A novel scanning system combined with specialized biochips enables detection down to individual fluorescent oligonucleotide molecules specifically hybridized to their complementary sequence over the entire biochip surface of cm2 size. A detection limit of 1.3 fM target oligonucleotide concentration—corresponding to only 39,000 molecules in the sample solution—and a dynamic range of 4.7 orders of magnitude have been achieved. The applicability of the system to PCR amplification-independent gene-expression profiling of minute samples was demonstrated by complex hybridization of cDNA derived from the equivalent of only 104 cells, which matches results obtained in ensemble studies on large samples. By counting each hybridized molecule on the microarray, the method is insusceptible to gene-specific variations of the labeling, thereby representing a principle advance to conventional ensemble-based microarray analysis.

We report here the development of a device for single-molecule imaging on large surface areas. A CCD camera operated in time delay and integration mode is synchronized with the movement of a sample scanning stage, enabling continuous data acquisition. Experiments on single fluorescent lipid molecules in supported lipid bilayers and on stained living cells demonstrate the capabilities of the method. Areas of up to 5 x 5 mm(2) were recorded within 11 min at a pixel size of 129 nm.