Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope''s objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.Download video file.^{(65M, mp4)}     

Beginning at the end: DNA replication within the telomere

Using single molecule analysis of replicated DNA (SMARD), Drosopoulos et al. (2015; J. Cell Biol. http://dx.doi.org/10.1083/jcb.201410061) report that DNA replication initiates at measurable frequency within the telomere of mouse chromosome arm 14q. They demonstrate that resolution of G4 structures on the G-rich template strand of the telomere requires some overlapping functions of BLM and WRN helicase for leading strand synthesis.Double-strand breaks in DNA can wreak havoc in cells if not repaired. Therefore, it was proposed that the ends of chromosomes may be specialized cap structures that are not recognized as double-strand breaks, thus preventing cell cycle arrest, degradation, and recombinational fusion (Muller, 1938; McClintock, 1939). We now know that telomeres comprise the ends of chromosomes and are essential for genome stability. Telomeres are composed of tandem head-to-tail repeats of a short G-rich sequence; for example, human telomeres are 2–20 kb of (TTAGGG)_n repeats. The chromosome ends are not blunt, and the 3′ end (G-rich strand) overhangs in a single strand that can invade the interior of the telomere to displace the internal G-rich sequence and form a T-loop structure (Griffith et al., 1999; Cesare et al., 2003; Doksani et al., 2013), thus protecting the chromosome ends from being recognized by the cell as double-strand breaks, in addition to protection by proteins that bind the telomere.Eukaryotic chromosomes are duplicated via semiconservative replication with a leading (continuous synthesis for net growth at the 3′ end of the nascent leading strand) and lagging (discontinuous Okazaki fragment synthesis for net growth at the 5′ end of the nascent lagging strand) elongating strand as shown in Fig. 1. In chromosomal semiconservative replication, the short 5′ RNA primer is removed from the nascent strand and the gap is filled in by DNA that is ligated to the adjacent nascent DNA. However, at the end of the chromosome, the gap after removal of the 5′ terminal RNA primer on the lagging strand cannot be filled in, and the chromosome may become shorter with each ensuing round of replication. This has been termed the end-replication problem (Watson, 1972; Olovnikov, 1973), and telomerase helps to solve this problem (Greider and Blackburn, 1987; Soudet et al., 2014).Open in a separate windowFigure 1.DNA replication at the end of chromosomes. (A) DNA replication can initiate within the subtelomeric region with replication forks (green arrows) progressing bidirectionally away from the origin. Telomere DNA is replicated by a replication fork that passes through this region. In each panel, leading nascent strand synthesis is indicated by a blue line with a single arrowhead; lagging nascent strand synthesis is indicated by a blue line with multiple arrowheads. At the top of each panel, the red line indicates the signal seen by microscopy of replication that initiated and continued during administration of the first pulse (IdU, red), and the dotted green line indicates the signal seen for replication extension during the second pulse (CldU, green). (B) On some DNA molecules from mouse chromosome 14q, DNA replication initiates within the telomere itself. In practice, the second (green) pulse was often not observed in the telomere. (C) Partially overlapping functions of BLM and WRN helicases are used to resolve G-quadruplex (G4) DNA (blue structure) that can form on the G-rich parental strand of the telomeres. In cells deficient of BLM and/or WRN helicase, progression of the nascent leading strand in the telomere is impaired; the slowed replication forks are indicated by red arrows. The resulting replication stress is accompanied by activation of dormant replication origins in the subtelomere. The cartoon is not drawn to scale, and the infrequently used subtelomeric replication origin in C is closer to the telomere than the subtelomeric origin in A.Semiconservative replication occurs before the action of telomerase. Previously it was thought that DNA replication began at an origin in chromosomal DNA adjacent to the telomere repeats, with the replication forks moving bidirectionally away from the subtelomeric origin (Fig. 1 A), thus replicating the telomere. However, the question remained whether DNA replication might initiate with some frequency within the telomere itself (Fig. 1 B). This question has now been answered in the affirmative in this issue by Drosopoulos et al., who used single molecule analysis of replicated DNA (SMARD; Norio and Schildkraut, 2001). In this approach, replicating cells are sequentially labeled by two different nucleotide analogues that are subsequently identified by immunofluorescence. For example, in bidirectional replication, red signals from the first pulse will be flanked at each end by green signals from the second pulse. Earlier reports using SMARD had concluded that most replication initiates at subtelomeric regions in the mouse and human genome and rarely in the telomeres themselves (Sfeir et al., 2009; Drosopoulos et al., 2012). In the recent study by Drosopoulos et al. (2015), fluorescence in situ hybridization (FISH) using probes from the telomere region allowed the replication pattern to be analyzed for a 320 kb genomic segment from the end of mouse chromosome arm 14q. Due to the long time (4 h) for the first (red) pulse, usually only red tracts of signal within the telomere were seen, but since many such molecules did not have the red signal extend into the subtelomeric region, it can be comfortably concluded that replication must have initiated within the telomere (Fig. 1 B). Moreover, some molecules did have red signal in the telomere flanked by green signal, supporting this conclusion. Although in these cases there was chromosome-proximal green signal, chromosome-distal green signal was rarely seen. Thus, although there was limited evidence for bidirectional replication originating in the telomere, it is very clear that a replication origin can exist within the telomere proper with a replication fork that extends over time into the subtelomere. It remains to be investigated whether replication initiates at a relatively high frequency in the telomeres of chromosomes other than 14q.These findings raise the question of whether the origin for DNA replication coincides with the simple sequence repeat found in telomeres or instead if it coincides with some other sequence that might be interspersed within the telomere. The former is suggested by a study with Xenopus cell-free extracts that could assemble the pre-replication complex and undergo some DNA replication on exogenous DNA containing exclusively telomeric repeats (Kurth and Gautier, 2010). Similar conclusions that DNA replication can initiate in the simple DNA repeats found in centromeres where replication bubbles have been observed in Drosophila virilis by electron microscopy have been reached (Zakian, 1976), and a recent study suggests that DNA replication initiates within human alpha-satellite DNA (Erliandri et al., 2014).Replications forks move slowly through telomeric DNA (Ivessa et al., 2002; Makovets et al., 2004; Miller et al., 2006; Sfeir et al., 2009) due to the high thermal stability of GC-rich telomeric DNA as well as its propensity to form stable secondary structures, such as G-quadruplex (G4) DNA, which can pose problems for DNA replication (Lopes et al., 2011; Paeschke et al., 2011). Various helicases help solve this problem; for example, Pif1 helicase helps to unwind G4 (Paeschke et al., 2013). Bloom syndrome helicase (BLM) and the Werner syndrome helicase (WRN) have also been implicated in assisting telomere replication: BLM suppresses replication-dependent fragile telomeres (Sfeir et al., 2009), and WRN suppresses defects in telomere lagging strand synthesis (Crabbe et al., 2004). Drosopoulos et al. (2015) now report that leading strand synthesis that initiates within the telomere has a slower rate of progression into the subtelomere in BLM-deficient cells as visualized by SMARD. Moreover, there was a higher frequency of replication initiation in the 14q subtelomere of the BLM-deficient cells, originating closer to the telomere than in BLM-proficient cells. These observations suggest that dormant replication origins in the 14q subtelomere can be activated when fork progression is impeded in BLM-deficient cells (Fig. 1 C). Drosopoulos et al. (2015) also found an increase in subtelomeric replication initiation when replication fork progression from the telomere was hindered by aphidicolin, as an alternate means to activate dormant origins by replication stress. When cells were treated with the G4 stabilizer PhenDC3, 14q subtelomeric origin firing increased further in BLM-deficient cells. Collectively, the data suggest a slowdown of progression of leading strand synthesis from an origin in the 14q telomere (using the G-rich parental strand as the template) when G4 structures cannot be resolved in BLM-deficient cells. As further support for a role of BLM helicase to remove G4 structures, there was increased staining in BLM-deficient cells by the BG4 antibody (Biffi et al., 2013) against G4 in the whole genome and especially in telomeres.WRN helicase can unwind G4 in vitro (Fry and Loeb, 1999; Mohaghegh et al., 2001). When Drosopoulos et al. (2015) used SMARD to analyze replication in cells doubly deficient of both BLM and WRN, they found a marked decrease of red replication signal in 14q telomeres, suggesting some functional overlap between BLM and WRN with regard to leading strand synthesis off the G-rich strand of telomeres. Supporting this conclusion, there was more G4 staining by the BG4 antibody in cells doubly deficient of both BLM and WRN than in cells deficient of just BLM or just WRN. This is the first direct demonstration in vivo of a contribution of BLM and WRN helicases in the resolution of G4 structures, which is especially needed for progression of leading strand synthesis that initiates in telomeres and is copied from the G-rich strand.     

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 ^1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins ³.Download video file.^{(133M, mp4)}     

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.Download video file.^{(82M, mov)}     

Break-Induced DNA Replication

Recombination-dependent DNA replication, often called break-induced replication (BIR), was initially invoked to explain recombination events in bacteriophage but it has recently been recognized as a fundamentally important mechanism to repair double-strand chromosome breaks in eukaryotes. This mechanism appears to be critically important in the restarting of stalled and broken replication forks and in maintaining the integrity of eroded telomeres. Although BIR helps preserve genome integrity during replication, it also promotes genome instability by the production of loss of heterozygosity and the formation of nonreciprocal translocations, as well as in the generation of complex chromosomal rearrangements.The break-copy mode of recombination (as opposed to break-join), was initially proposed by Meselson and Weigle (1961). Break-copy recombination, now more commonly known as recombination-dependent DNA replication or break-induced replication (BIR), is believed to account for restarting replication at broken replication forks and may also play a central role in the maintenance of telomeres in the absence of telomerase. BIR has been studied in various model systems and has been invoked to explain chromosome rearrangements in humans. This review focuses primarily on mechanistic studies in Escherichia coli and its bacteriophages, T4 and λ, in the budding yeasts Saccharomyces cerevisiae and Kluyveromyces lactis and on apparently similar, but less well-documented, mechanisms in mammalian cells.Homology-dependent repair of DNA double-strand breaks (DSBs) occur by three major repair pathways (Pâques and Haber 1999) (Fig. 1). When both ends of the DNA share substantial homology with a donor template (a sister chromatid, a homologous chromosome, or an ectopically located segment), repair occurs almost exclusively by gene conversion (GC). If the DSB is flanked by direct repeats, then a second repair process, single-strand annealing (SSA), can occur as 5′ to 3′ resection of the DSB ends exposes complementary sequences that can anneal to each other and repair the break by the formation of a deletion. However, when only one DSB end shares homology with a donor sequence, repair occurs by BIR. There are two BIR pathways, one dependent on Rad51 recombinase and the other independent of Rad51.Open in a separate windowFigure 1.Three major repair pathways of homology-dependent recombination. Noncrossover (NCO) and crossover (CO) events are indicated. Black triangles represent resolution of Holliday junctions (HJs). Dashed lines represent new DNA synthesis. GC, gene conversion; SSA, single-strand annealing; BIR, break-induced replication.     

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes.Download video file.^{(62M, mov)}     

Manipulation of single polymerase-DNA complexes: A mechanical view of DNA unwinding during replication

Comment on: Morin JA, et al. Proc Natl Acad Sci USA 2012; 109:8115-20.DNA replication requires overcoming the energetic barrier associated with the base pair melting of its double helix and a fine-tuned coordination between the processes of DNA unwinding and DNA replication. One intriguing question that remains poorly understood is the exact mechanism of the coupling of these two reactions. In some organisms, these activities are coupled within the same protein, like in the case of the phage Phi29 DNA polymerase. This polymerase works as a hybrid polymerase-helicase, because it presents an amino acid insertion that together with other protein domains forms a narrow tunnel around the template strand. This topological restriction is similar to the one imposed by hexameric helicases at the fork junction and promotes the separation of the fork ahead.¹ The Phi29 DNA polymerase, therefore, constitutes a simple, good model system to understand the basic mechanistic principles of the coupling between DNA replication and unwinding activities: the polymerase may behave as a “passive” unwinding motor, if translocation of the protein traps transient unwinding fluctuations of the fork, or as an “active” motor, if the polymerase actively destabilizes the duplex DNA at the junction. Therefore, factors that affect the stability of the fork junction, as DNA sequence or mechanical destabilization of the fork, will have a stronger effect on the unwinding kinetics of a “passive” motor than on an “active” one.To determine the DNA unwinding mechanism of the Phi29 DNA polymerase, we used optical tweezers to measure at single molecule level the effect of DNA sequence and destabilizing forces on the fork on the rates of strand displacement (replication and unwinding are tightly coupled, Δx₁, Fig. 1A) and primer extension (replication of the displaced complementary strand without unwinding, Δx₂, Fig. 1A) of two polymerases: the wild-type Phi29 DNA polymerase and a strand displacement deficient variant, which bears a couple of mutations that may affect the stability of the tunnel required for unwinding.² We quantified the free energy of interaction between the polymerase and the DNA fork, ΔG_int, and the range of this interaction, M, through a theoretical analysis of the dependence of the replication, unwinding and pause kinetics on the DNA sequence and force.^3,4Open in a separate windowFigure 1. (A) Schematic representation of the experimental design (not to scale). A single DNA hairpin was attached to functionalized beads inside a fluidics chamber. One strand of the hairpin is attached through a dsDNA handle to a bead held in the optical trap (top), while the complementary strand is attached to a bead on top of a mobile micropipette (bottom). At a constant force, after flowing the nucleotides into the reaction chamber, the strand displacement and primer extension rates of the polymerase are detected as a change in distance between the beads, Δx₁ and Δx₂, respectively. (B) Representative replication activity of a single mutant polymerase molecule. Long pauses are observed only during the strand displacement reaction. (C) Mechanistic distinction between passive and active unwinding. The cartoon illustrates the degree of activeness in DNA unwinding of different replicative helicases⁶ and the Phi29 DNA polymerase.Our results show that while the primer extension rates of both polymerases are force- and sequence-independent their average unwinding rates are sensitive to these two variables, although with different intensity. As expected, the dsDNA fork presents a much stronger physical barrier to the mutant polymerase unwinding. Qualitative reasoning might suggest that the observed differences imply different “activeness” of the unwinding mechanism of each polymerase. However, the inclusion of the pause kinetics of each polymerase in our model revealed that they use the same active mechanism; they both destabilize the two nearest base pairs of the fork (M = 2) with an interaction energy ΔG_int = 2 k_BT per base pair. These results suggest that mutations affecting the stability of the tunnel required for unwinding do not decrease the “activeness” of the motor but instead increase the probability of the unwinding mechanism to fail upon encountering a closed fork junction, inducing the entrance of the mutant polymerase into a long-lived inactive pause state (Fig. 1B). These results bring out the importance to consider pause kinetics to accurately quantify the actual unwinding mechanism of the Phi29 DNA polymerase or any other nucleic acid unwinding motor in which pauses are relevant during its operation. The presence of pauses obscures the actual pause-free rates of the motor and can lead to misleading results when they are not properly accounted.Our data are consistent with a model in which the closed template tunnel that wraps around the template strand allows the Phi29 DNA polymerase to maintain a sharp bending of this strand (essential for template reading in all replicative polymerases) and a bending of the complementary strand, due to its steric exclusion, at a closed fork junction. Bending of the two strands would generate mechanical stress at the junction promoting its active destabilization. A less stable tunnel, as in the mutant polymerase, will not be able to keep the mechanical stress at a closed fork junction, in this case the fork pressure would induce loosening of the correct protein-DNA interactions favoring the entrance to a polymerization inactive state.Similar mechanisms for mechanical destabilization of the fork junction can be envisioned for other DNA replication systems in which a DNA polymerase and a helicase work in coordination. In these systems, the leading strand can be sharply bent by the steric exclusion induced by the helicase and by the functional binding of the polymerase generating effective mechanical stress at the fork junction to account for efficient unwinding during replication. These implications are further supported by recent single molecule studies using magnetic tweezers that describe a collaborative coupling of this nature between the activities of the bacteriophage T4 DNA polymerase and DNA helicase.⁵     

From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated.Open in a separate windowClick here to view.^{(24M, flv)}     

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.Open in a separate windowClick here to view.^{(28M, flv)}     

DNA Stable-Isotope Probing (DNA-SIP)

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.Download video file.^{(159M, mp4)}     

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.Download video file.^{(51M, flv)}     

Splenogonadal Fusion: An Unusual Case of an Acute Scrotum

We highlight a case on a normal left testicle with a fibrovascular cord with three nodules consistent with splenic tissue. The torsed splenule demonstrated hemorrhage with neutrophilic infiltrate and thrombus consistent with chronic infarction and torsion. Splenogonadal fusion (SGF) is a rather rare entity, with approximately 184 cases reported in the literature. The most comprehensive review was that of 123 cases completed by Carragher in 1990. Since then, an additional 61 cases have been reported in the scientific literature. We have studied these 61 cases in detail and have included a summary of that information here.Key words: Splenogonadal fusion, Acute scrotumA 10-year-old boy presented with worsening left-sided scrotal pain of 12 hours’ duration. The patient reported similar previous episodes occurring intermittently over the past several months. His past medical history was significant for left hip dysplasia, requiring multiple hip surgeries. On examination, he was found to have an edematous left hemiscrotum with a left testicle that was rigid, tender, and noted to be in a transverse lie. The ultrasound revealed possible polyorchism, with two testicles on the left and one on the right (Figure 1), and left epididymitis. One of the left testicles demonstrated a loss of blood flow consistent with testicular torsion (Figure 2).Open in a separate windowFigure 1Ultrasound of the left hemiscrotum reveals two spherical structures; the one on the left is heterogeneous and hyperdense in comparison to the right.Open in a separate windowFigure 2Doppler ultrasound of left hemiscrotum. No evidence of blood flow to left spherical structure.The patient was taken to the operating room for immediate scrotal exploration. A normalappearing left testicle with a normal epididymis was noted. However, two accessory structures were noted, one of which was torsed 720°; (Figure 3). An inguinal incision was then made and a third accessory structure was noted. All three structures were connected with fibrous tissue, giving a “rosary bead” appearance. The left accessory structures were removed, a left testicular biopsy was taken, and bilateral scrotal orchipexies were performed.Open in a separate windowFigure 3Torsed accessory spleen with splenogonadal fusion.Pathology revealed a normal left testicle with a fibrovascular cord with three nodules consistent with splenic tissue. The torsed splenule demonstrated hemorrhage with neutrophillic infiltrate and thrombus consistent with chronic infarction and torsion (Figure 4).Open in a separate windowFigure 4Splenogonadal fusion, continuous type with three accessory structures.     

DNA Replication Origins

The onset of genomic DNA synthesis requires precise interactions of specialized initiator proteins with DNA at sites where the replication machinery can be loaded. These sites, defined as replication origins, are found at a few unique locations in all of the prokaryotic chromosomes examined so far. However, replication origins are dispersed among tens of thousands of loci in metazoan chromosomes, thereby raising questions regarding the role of specific nucleotide sequences and chromatin environment in origin selection and the mechanisms used by initiators to recognize replication origins. Close examination of bacterial and archaeal replication origins reveals an array of DNA sequence motifs that position individual initiator protein molecules and promote initiator oligomerization on origin DNA. Conversely, the need for specific recognition sequences in eukaryotic replication origins is relaxed. In fact, the primary rule for origin selection appears to be flexibility, a feature that is modulated either by structural elements or by epigenetic mechanisms at least partly linked to the organization of the genome for gene expression.Timely duplication of the genome is an essential step in the reproduction of any cell, and it is not surprising that chromosomal DNA synthesis is tightly regulated by mechanisms that determine precisely where and when new replication forks are assembled. The first model for a DNA synthesis regulatory circuit was described about 50 years ago (Jacob et al. 1963), based on the idea that an early, key step in building new replication forks was the binding of a chromosomally encoded initiator protein to specialized DNA regions, termed replication origins (Fig. 1). The number of replication origins in a genome is, for the most part, dependent on chromosome size. Bacterial and archaeal genomes, which usually consist of a small circular chromosome, frequently have a single replication origin (Barry and Bell 2006; Gao and Zhang 2007). In contrast, eukaryotic genomes contain significantly more origins, ranging from 400 in yeast to 30,000–50,000 in humans (Cvetic and Walter 2005; Méchali 2010), because timely duplication of their larger linear chromosomes requires establishment of replication forks at multiple locations. The interaction of origin DNA and initiator proteins (Fig. 1) ultimately results in the assembly of prereplicative complexes (pre-RCs), whose role is to load and activate the DNA helicases necessary to unwind DNA before replication (Remus and Diffley 2009; Kawakami and Katayama 2010). Following helicase-catalyzed DNA unwinding, replisomal proteins become associated with the single-stranded DNA, and new replication forks proceed bidirectionally along the genome until every region is duplicated (for review, see O’Donnell 2006; Masai et al. 2010).Open in a separate windowFigure 1.Revised versions of the replicon model for all domains of life. For cells of each domain type, trans-acting initiators recognize replication origins to assemble prereplicative complexes required to unwind the DNA and load DNA helicase. Eukaryotic initiators are preassembled into hexameric origin recognition complexes (ORCs) before interacting with DNA. In prokaryotes, single initiators (archaeal Orc1/Cdc6 or bacterial DnaA) bind to recognition sites and assemble into complexes on DNA. In all cases, the DNA helicases (MCMs or DnaB) are recruited to the origin and loaded onto single DNA strands. In bacteria, DNA-bending proteins, such as Fis or IHF, may modulate the assembly of pre-RC by bending the origin DNA. Two activities of DnaA are described in the figure. The larger version binds to recognition sites, and the smaller version represents DnaA required to assist DnaC in loading DnaB helicase on single-stranded DNA.Initiator proteins from all forms of life share structural similarities, including membership in the AAA⁺ family of proteins (ATPases associated with various cellular activities) (Duderstadt and Berger 2008; Wigley 2009) that are activated by ATP binding and inactivated by ATP hydrolysis (Duderstadt and Berger 2008; Duncker et al. 2009; Kawakami and Katayama 2010). Despite these similarities, initiators assemble into prereplicative complexes in two fundamentally different ways (Fig. 2). In prokaryotes, initiator monomers interact with the origin at multiple repeated DNA sequence motifs, and the arrangement of these motifs (see below) can direct assembly of oligomers that mediate strand separation (Erzberger et al. 2006; Rozgaja et al. 2011). In eukaryotes, a hexameric origin recognition complex (ORC) binds to replication origins and then recruit additional factors (as Cdc6 and Cdt1) that will themselves recruit the hexameric MCM2-7 DNA helicase to form a prereplicative complex (for review, see Diffley 2011). This process occurs during mitosis and along G₁ and is called “DNA replication licensing,” a crucial regulation of eukaryotic DNA replication (for review, see Blow and Gillespie 2008). Importantly, this complex is still inactive, and only a subset of these preassembled origins will be activated in S phase. This process is, therefore, fundamentally different from initiation of replication in bacteria. Moreover, because sequence specificity appears more relaxed in large eukaryotic genomes, prokaryotic mechanisms that regulate initiator–DNA site occupation must be replaced by alternative mechanisms, such as structural elements or the use of epigenetic factors.Open in a separate windowFigure 2.Functional elements in some well-studied prokaryotic replication origins. (A) Bacterial oriCs. The DNA elements described in the text are (arrows) DnaA recognition boxes or (boxes) DNA unwinding elements (DUEs). When recognition site affinities are known, colored arrows designate high- (K_d > 100 nm) and low- (K_d < 100 nm) affinity sites. (B) Archaeal oriCs. Arrows and boxes designate DNA elements as in A, but the initiator protein is Orc1/Cdc6 rather than DnaA. (Thick arrows) Long origin recognition boxes (ORBs); (thin arrows) shorter versions (miniORBs). Both ORBs and miniORBs are identified in Pyrococcus. DUEs are not yet well defined for Helicobacter or Sulfolobus genera and are not labeled in this figure.Here, we describe replication origins on prokaryotic and eukaryotic genomes below, with a particular focus on the attributes responsible for orderly initiator interactions and origin selection specificity, as well as on the shift from origin sequence-dependent regulation to epigenetic regulation. You are also referred to other related articles in this collection and several recent reviews covering the topics of DNA replication initiation in more detail (Méchali 2010; Beattie and Bell 2011; Blow et al. 2011; Bryant and Aves 2011; Ding and MacAlpine 2011; Dorn and Cook 2011; Kaguni 2011; Leonard and Grimwade 2011; Sequeira-Mendes and Gomez 2012).     

Protocols for Oral Infection of Lepidopteran Larvae with Baculovirus

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This video shows how lepidopteran larvae can be infected with polyhedra by droplet feeding and diet plug-based bioassays. This accompanying Springer Protocols section provides an overview of the baculovirus lifecycle and use of baculoviruses as insecticidal agents, including discussion of the pros and cons for use of baculoviruses as insecticides, and progress made in genetic enhancement of baculoviruses for improved insecticidal efficacy.Open in a separate windowClick here to view.^{(52M, flv)}     

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.Open in a separate windowClick here to view.^{(19M, flv)}