Enhancement of Escherichia Coli Plasmid and Chromosomal Recombination by the Ref Function of Bacteriophage P1

The Ref activity of phage P1 enhances recombination between two defective lacZ genes in the Escherichia coli chromosome (lac- x lac- recombination). Plasmid recombination, both lac- x lac- and tet- x tet-, was measured by transformation of recA strains, and was also assayed by measurement of beta-galactosidase. The intracellular presence of recombinant plasmids was verified directly by Southern blotting. Ref stimulated recombination of plasmids in rec+ and rec(BCD) cells by 3-6-fold, and also the low level plasmid recombination in recF cells. RecA-independent plasmid recombination, either very low level (recA cells) or high level (recB recC sbcA recA cells), was not stimulated. Ref stimulated both intramolecular and intermolecular plasmid recombination. Both normal and Ref-stimulated lac- x lac- chromosomal recombination, expected to be mostly RecBC-dependent in wild-type bacteria, were affected very little by a recF mutation. We have previously reported Ref stimulation of lac- x lac- recombination in recBC sbcB bacteria, a process known to be RecF-dependent. Chromosomal recombination processes thought to involve activated recombination substrates, e.g., Hfr conjugation, P1 transduction, were not elevated by Ref activity. We hypothesize that Ref acts by unknown mechanisms to activate plasmid and chromosomal DNA for RecA-mediated recombination, and that the structures formed are substrates for both RecF-dependent (plasmid, chromosomal) and Rec(BCD)-dependent (chromosomal) recombination pathways.     

Binding of ColE1-kan Plasmid DNA by Tobacco Protoplasts: Nonexpression of Plasmid Gene

Protoplasts prepared from cultured tobacco cells were treated with ColE1-kan plasmid DNA, a hybrid of ColE1 and pSC105 plasmids bearing a gene for kanamycin resistance. The conditions employed permitted the uptake or irreversible binding of 2.9% of the added DNA in acid-insoluble form. Upon commencement of division, the treated cells were plated in agar medium containing kanamycin and differentiating hormones. Plantlets or shoots obtained as presumptive transformants were further tested on kanamycin medium by subculturing small leaf pieces. No evidence was obtained for expression of the kanamycin resistance gene of ColE1-kan in tobacco tissue.     

Hop1: A Yeast Meiotic Pairing Gene

The recessive mutation, hop1-1, was isolated by use of a screen designed to detect mutations defective in homologous chromosomal pairing during meiosis in Saccharomyces cerevisiae. Mutants in HOP1 displayed decreased levels of meiotic crossing over and intragenic recombination between markers on homologous chromosomes. In contrast, assays of the hop1-1 mutation in a spo13-1 haploid disomic for chromosome III demonstrated that intrachromosomal recombination between directly duplicated sequences was unaffected. The spores produced by SPO13 diploids homozygous for hop1 were largely inviable, as expected for a defect in interhomolog recombination that results in high levels of nondisjunction. HOP1 was cloned by complementation of the spore lethality phenotype and the cloned gene was used to map HOP1 to the LYS11-HIS6 interval on the left arm of chromosome IX. Electron microscopy revealed that diploids homozygous for hop1 fail to form synaptonemal complex, which normally provides the structural basis for homolog pairing. We propose that HOP1 acts in meiosis primarily to promote chromosomal pairing, perhaps by encoding a component of the synaptonemal complex.     

Switching Protein-DNA Recognition Specificity by Single-Amino-Acid Substitutions in the P1 par Family of Plasmid Partition Elements

The P1, P7, and pMT1 par systems are members of the P1 par family of plasmid partition elements. Each has a ParA ATPase and a ParB protein that recognizes the parS partition site of its own plasmid type to promote the active segregation of the plasmid DNA to daughter cells. ParB contacts two parS motifs known as BoxA and BoxB, the latter of which determines species specificity. We found that the substitution of a single orthologous amino acid in ParB for that of a different species has major effects on the specificity of recognition. A single change in ParB can cause a complete switch in recognition specificity to that of another species or can abolish specificity. Specificity changes do not necessarily correlate with changes in the gross DNA binding properties of the protein. Molecular modeling suggests that species specificity is determined by the capacity to form a hydrogen bond between ParB residue 288 and the second base in the BoxB sequence. As changes in just one ParB residue and one BoxB base can alter species specificity, plasmids may use such simple changes to evolve new species rapidly.     

A Role for the CAL1-Partner Modulo in Centromere Integrity and Accurate Chromosome Segregation in Drosophila

The relationship between the nucleolus and the centromere, although documented, remains one of the most elusive aspects of centromere assembly and maintenance. Here we identify the nucleolar protein, Modulo, in complex with CAL1, a factor essential for the centromeric deposition of the centromere-specific histone H3 variant, CID, in Drosophila. Notably, CAL1 localizes to both centromeres and the nucleolus. Depletion of Modulo, by RNAi, results in defective recruitment of newly-synthesized CAL1 at the centromere. Furthermore, depletion of Modulo negatively affects levels of CID at the centromere and results in chromosome missegregation. Interestingly, examination of Modulo localization during mitosis reveals it localizes to the chromosome periphery but not the centromere. Combined, the data suggest that rather than a direct regulatory role at the centromere, it is the nucleolar function of modulo which is regulating the assembly of the centromere by directing the localization of CAL1. We propose that a functional link between the nucleolus and centromere assembly exists in Drosophila, which is regulated by Modulo.     

Improved Transient Protein Expression by pFluNS1 Plasmid

Influenza virus nonstructural protein-1 (NS1) is abundantly expressed in influenza virus infected cells. NS1 is well recognized for counteracting host antiviral activities and regulating host and viral protein expression. When used as a plasmid component in DNA transfection, NS1 was shown to significantly increase expression levels of a cotransfected gene of different plasmid. Our previous studies demonstrated that addition of an NS1 plasmid increased the expression levels of influenza virus secreted neuraminidase (sNA) gene in 293T cells. In this study, we improved the utilization of NS1 as an enhancer for transient protein expression by generating pFluNS1 plasmid to contain two expression cassettes; one encoding an NS1 gene and another encoding a gene of interest. pFluNS1 is expected to codeliver the NS1 gene into the same cells receiving the gene of interest. The plasmid is therefore designed to induce higher protein expression levels than a cotransfection of an NS1 plasmid and a plasmid containing a gene of interest. To test the efficiency of pFluNS1, influenza virus sNA and non-viral DsRed genes were cloned into pFluNS1. The expression of these genes from pFluNS1 was then compared to the expression from a cotransfection of an NS1 plasmid and an expression plasmid coding for sNA or DsRed. We found that gene expression from pFluNS1 reached equal or higher levels to those derived from the cotransfection. Because the expression from pFluNS1 needs only one plasmid, a lesser amount of transfection reagent was required. Thus, the use of pFluNS1 provides a transfection approach that reduces the cost of protein expression without compromising high levels of protein expression. Together, these data suggest that pFluNS1 can serve as a novel alternative for an efficient transient protein expression in mammalian cells.     

Plasmid P1 RepA is homologous to the F plasmid RepE class of initiators

DNA replication of plasmid P1 requires a plasmid-encoded origin DNA-binding protein, RepA. RepA is an inactive dimer and is converted by molecular chaperones into an active monomer that binds RepA binding sites. Although the sequence of RepA is not homologous to that of F plasmid RepE, we found by using fold-recognition programs that RepA shares structural homology with RepE and built a model based on the RepE crystal structure. We constructed mutants in the two predicted DNA binding domains to test the model. As expected, the mutants were defective in P1 DNA binding. The model predicted that RepA binds the first half of the binding site through interactions with the C-terminal DNA binding domain and the second half through interactions with the N-terminal domain. The experiments supported the prediction. The model was further supported by the observation that mutants defective in dimerization map to the predicted subunit interface region, based on the crystal structure of pPS10 RepA, a RepE family member. These results suggest P1 RepA is structurally homologous to plasmid initiators, including those of F, R6K, pSC101, pCU1, pPS10, pFA3, pGSH500, Rts1, RepHI1B, RepFIB, and RSF1010.     

The Structure and Assembly Dynamics of Plasmid Actin AlfA Imply a Novel Mechanism of DNA Segregation

Bacterial cytoskeletal proteins participate in a variety of processes, including cell division and DNA segregation. Polymerization of one plasmid-encoded, actin-like protein, ParM, segregates DNA by pushing two plasmids in opposite directions and forms the current paradigm for understanding active plasmid segregation. An essential feature of ParM assembly is its dynamically instability, the stochastic switching between growth and disassembly. It is unclear whether dynamic instability is an essential feature of all actin-like protein-based segregation mechanisms or whether bacterial filaments can segregate plasmids by different mechanisms. We expressed and purified AlfA, a plasmid-segregating actin-like protein from Bacillus subtilis, and found that it forms filaments with a unique structure and biochemistry; AlfA nucleates rapidly, polymerizes in the presence of ATP or GTP, and forms highly twisted, ribbon-like, helical filaments with a left-handed pitch and protomer nucleotide binding pockets rotated away from the filament axis. Intriguingly, AlfA filaments spontaneously associate to form uniformly sized, mixed-polarity bundles. Most surprisingly, our biochemical characterization revealed that AlfA does not display dynamic instability and is relatively stable in the presence of diphosphate nucleotides. These results (i) show that there is remarkable structural diversity among bacterial actin filaments and (ii) indicate that AlfA filaments partition DNA by a novel mechanism.Bacteria contain multiple filament-forming proteins related to eukaryotic actin (6). These actin-like proteins have multiple cellular roles, including determination of cell shape (18), arrangement of organelles (20), and segregation of DNA (36). Little is known about the assembly dynamics of most of these proteins or about the identities and activities of the factors that regulate them. The widely expressed actin-like protein MreB, for example, has been purified and studied in vitro, but its assembly appears to be strongly inhibited by physiological concentrations of monovalent cations, suggesting that its assembly in vivo is facilitated by as-yet-unknown factors (23). At present, the best-understood actin-like protein is ParM, a plasmid-encoded protein that constructs a bipolar spindle capable of pushing plasmids to opposite poles of rod-shaped cells (5, 25). In contrast to the eukaryotic actin cytoskeleton, whose assembly and architecture are regulated by a variety of accessory factors, ParM dynamics are regulated by a single factor, a complex composed of multiple copies of the repressor protein ParR bound to a DNA locus, parC (17). The ParR/parC complex binds the ends of ParM filaments and is pushed through the cytoplasm by filament elongation (5, 14, 25). The ability of ParM to function with such minimal regulation appears to be due to its unique assembly dynamics, which are dramatically different from those of eukaryotic actins. One of the most important differences is that ParM filaments are dynamically unstable (13). That is, similar to eukaryotic microtubules, they can exist in one of two states: stably growing or rapidly (catastrophically) shrinking. This property is required for the ability of ParM to segregate DNA in vivo and appears to solve several fundamental problems associated with DNA segregation. First, spontaneous disassembly of the polymer overcomes the need for an accessory factor to take filaments apart. Second, because filaments bound to ParR/parC complexes are selectively stabilized, the catastrophic disassembly of unattached filaments provides excess monomers that can preferentially elongate them. This is significant because, if the stabilities of attached and unattached filaments were similar, the concentration of free ParM monomers would equilibrate at a level not capable of promoting DNA segregation. And finally, pairs of plasmids appear to find each other via a search-and-capture mechanism (5, 14) that is dramatically enhanced by the continual growth and shortening of filaments attached to single plasmids (16).Because we have little information on the dynamics of other actin-like proteins, it is unclear to what extent ParM''s behavior reflects general properties of bacterial actins rather than specific adaptations to its role in DNA segregation. Furthermore, it is unclear whether all plasmid-segregating actins employ the same dynamic instability-based strategy to find and transport DNA molecules. To better understand the structural and functional diversity of bacterial actins, we studied a second, recently discovered plasmid-segregating actin-like protein, AlfA (1). The AlfA gene is part of an operon (alf) that is located close to the origin of replication of a ∼70-kb, low-copy-number plasmid, pLS32. This plasmid was initially isolated from a natto strain of Bacillus subtilis used in soybean fermentation (33), but a similar plasmid with an identical alf operon is also present in a colony-forming laboratory strain of B. subtilis, strain NCIB 3610 (8, 32). The function of these plasmids is cryptic. They are present at levels of only two or three copies per chromosome equivalent (33), and maintenance of their derivatives requires both AlfA and a downstream gene, alfB (1). Becker and coworkers (1) identified AlfA as a member of the actin superfamily based on the presence of a conserved nucleotide binding fold (4), although the sequence of AlfA is as different from the sequences of ParM and MreB as all three are from the sequence of conventional eukaryotic actin (∼20% identity). These authors also showed that fluorescent derivatives of AlfA form a single filamentous structure running along the long axis of the cell. Photobleached filaments recover from both ends in approximately 1 min, indicating that the structures are composed of multiple, dynamic filaments (1). By analogy with the ParR/parC complex, AlfB might be a DNA binding protein that couples AlfA assembly to plasmid movement. To date, no centromeric sequences involved in segregation have been identified in this plasmid.We expressed and purified AlfA and characterized its assembly dynamics by using light scattering, high-speed pelleting, and fluorescence microscopy, and we determined the structure of AlfA polymers by high-resolution electron microscopy (EM). We found that in the presence of ATP and GTP, AlfA forms two-strand helical filaments and filament bundles. Like ParM filaments, AlfA filaments are left-handed two-start helices, but otherwise their filament architecture is quite different. AlfA filaments appear to be more tightly twisted and ribbon-like, and AlfA subunits have a significantly different orientation with respect to the filament axis. Unlike other actin-like proteins described thus far, AlfA spontaneously forms regularly sized, mixed-polarity filament bundles driven by electrostatic interactions between filaments, even in the absence of molecular crowding. Finally, AlfA shows no evidence of the dynamic instability crucial to the function of ParM. Thus, AlfA assembles into a unique structure with a unique set of biochemical and structural properties, suggesting a novel mechanism for DNA segregation.     

Cloning and Characterization of theoriRegion of pSW1200 ofErwinia stewartii:Similarity with Plasmid P1

Theoriregion of anErwinia stewartiiplasmid, pSW1200 (106 kb), has been cloned and sequenced. This region consists of a gene encoding a protein which has 91% similarity and 73% identity with the RepA protein of bacteriophage P1. Theoriregion also consists of eight copies of 19-bp iterons which are highly homologous to the iterons of P1. Similar to plasmid P1, pSW1200 replicon has a copy number of approximately 1. On the other hand, the copy number increases about ninefold if three of the iterons located downstream fromrepAgene are deleted. We also demonstrate that pGEM-5Z consisting of a copy of P1 iteron is incompatible with a pSW1200 derivative, pSW1201, suggesting that pSW1200 and P1 DNA are incompatible and both belong to the IncY group.     

Asymmetric Segregation and Polarized Redistribution of Pole Plasm During Early Cleavages in the Tubifex Embryo: Role of Actin Networks and Mitotic Apparatus

In the precleavage zygote of Tubifex , pole plasm, which is yolk-free cytoplasm, is located at the animal and vegetal poles. The present study describes the fate and localization pattern of the pole plasms in embryonic development of Tubifex . The process of pole plasm localization during cleavage stages is comprised of three steps. The first step is asymmetric segregation which results in bipolar localization of pole plasm masses in the D-cell of the 4-cell embryo. The spatial organization of pole plasm at this stage depends on F-actin but not on microtubules. The second step is the redistribution of the vegetal pole plasm toward the animal pole and its unification with the animal pole plasm. These give rise to localization of unified pole plasm at the animal side (i.e. future dorsal side of the embryo) of the D-quadrant. The polarized redistribution is sensitive to colchicine and topographically related to the mitotic apparatus located at the animal pole of the D-cell. Electron microscopy shows the association with astral microtubules of constituents of pole plasm, suggesting the involvement of astral microtubules in cytoplasmic movement which gives rise to redistribution. In addition, centrifuge experiments suggest that the directional information for this polarized redistribution may be provided by some cytoplasmic organizations which are resistant to centrifugal force. The last step of the localization process is partitioning of unified pole plasm into two micromeres 2d and 4d. The spatial organization of pole plasm at this stage depends on microtubules but not on F-actin.     

Plasmid pVAX1-NH36 purification by membrane and bead perfusion chromatography

Bioprocess and Biosystems Engineering - The demand for plasmid DNA (pDNA) has increased in response to the rapid advances in vaccines applications to prevent and treat infectious diseases caused by...     

Plasmid pRTL1 Controlling 1-Chloroalkane Degradation by Rhodococcus rhodochrous NCIMB13064

Rhodococcus rhodochrous NCIMB13064 can dehalogenate and use a wide range of 1-haloalkanes as sole carbon and energy source. The 1-chloroalkane degradation phenotype may be lost by cells spontaneously or after treatment with Mitomycin C. Two laboratory derivatives of the original strain exhibited differing degrees of stability of the chloroalkane degradation marker. Plasmids of approximately 100 kbp (pRTL1) and 80 kbp (pRTL2) have been found in R. rhodochrous NCIMB13064. pRTL1 was shown to be carrying at least some genes for the dehalogenation of 1-chloroalkanes with short chain lengths (C₃ to C₉). However, no connection was found between the utilization of 1-chloroalkanes with longer chain lengths (C₁₂ to C₁₈) and the presence of pRTL1. Three separate events were observed to lead to the inability of NCIMB13064 to dehalogenate the short-chain 1-chloroalkanes; the complete loss of pRTL1, the integration of pRTL1 into the chromosome, or the deletion of a 20-kbp fragment in pRTL1. High-frequency transfer of the 1-chloroalkane degradation marker associated with pRTL1 has been demonstrated in bacterial crosses between different derivatives of R. rhodochrous NCIMB13064.     

S-Nitrosation of Glutathione Transferase P1-1 Is Controlled by the Conformation of a Dynamic Active Site Helix

S-Nitrosation is a post-translational modification of protein cysteine residues, which occurs in response to cellular oxidative stress. Although it is increasingly being linked to physiologically important processes, the molecular basis for protein regulation by this modification remains poorly understood. We used transient kinetic methods to determine a minimal mechanism for spontaneous S-nitrosoglutathione (GSNO)-mediated transnitrosation of human glutathione transferase (GST) P1-1, a major detoxification enzyme and key regulator of cell proliferation. Cys⁴⁷ of GSTP1-1 is S-nitrosated in two steps, with the chemical step limited by a pre-equilibrium between the open and closed conformations of helix α2 at the active site. Cys¹⁰¹, in contrast, is S-nitrosated in a single step but is subject to negative cooperativity due to steric hindrance at the dimer interface. Despite the presence of a GSNO binding site at the active site of GSTP1-1, isothermal titration calorimetry as well as nitrosation experiments using S-nitrosocysteine demonstrate that GSNO binding does not precede S-nitrosation of GSTP1-1. Kinetics experiments using the cellular reductant glutathione show that Cys¹⁰¹-NO is substantially more resistant to denitrosation than Cys⁴⁷-NO, suggesting a potential role for Cys¹⁰¹ in long term nitric oxide storage or transfer. These results constitute the first report of the molecular mechanism of spontaneous protein transnitrosation, providing insight into the post-translational control of GSTP1-1 as well as the process of protein transnitrosation in general.     

Redistribution of rat renal allograft-responding leukocytes during rejection: 1. Model

We describe a model that enables us to trace the traffic of allograft-responding inflammatory leukocytes to and from the graft without handling of these cells in vitro. At different times after transplantation, the kidney transplant pedicle—including the artery, vein, and draining lymphatics—is clamped. The allograft-responding leukocytes are labeled by a [³H]thymidine pulse either in situ or in the systemic lymphoid organs of the recipient. Fifteen minutes later the pulse is chased with a 1000-fold excess of cold thymidine, and the clamp is opened. The animals are sacrificed 18 hr later, when a balance between the synthesis of new labeled leukocytes from the originally labeled ones and dilution of intracellular label has been achieved. This model was used to analyze the allograft-responding inflammatory cell traffic to and from a renal transplant performed across the major histocompatibility complex in the rat. A sizable traffic was observed to both directions: After systemic injection of label only 0.008 × 10⁶ labeled cells × hr^?1 were found to emigrate into a kidney allograft (control). Already on the third day after transplantation—when the in situ inflammatory response is still at its beginning—more than 0.3 × 10⁶ labeled cells × hr^?1 migrated from the host to the allograft and 1.6 × 10⁶ labeled cells × hr^?1 left the allograft to the recipient spleen. The first figure is several-fold higher than any previous estimate. The findings emphasize the systemic nature of the antiallograft inflammatory response.