In vitro inhibition of fungal activity by macrophage-mediated sequestration and release of encapsulated amphotericin B nanosupension in red blood cells.

The efficacy of antifungal treatment has been diminished by the biodistribution limitations of amphotericin B (AmB) due to its pharmacological profile, as well as the severe side effects it causes. A cellular drug-delivery system, which incorporates human erythrocytes (RBCs) loaded with an AmB nanosuspension (AmB-NS), is developed in order to improve antifungal treatment. AmB-NS encapsulation in RBCs is achieved by using hypotonic hemolysis, leading to intracellular AmB amounts of 3.81 +/- 0.47 pg RBC(-1) and an entrapment efficacy of 15-18%. Upon phagocytosis of AmB-NS-RBCs, leukocytes show a slow AmB release over ten days, and no alteration in cell viability. This results in an immediate, permanent inhibition of intra- and extracellular fungal activity. AmB-NS-RBC-leukocyte-mediated delivery of AmB is efficient in amounts 1000 times lower than the toxic dose. This drug-delivery method is effective for the transport of water-insoluble substances, such as AmB, and this warrants consideration for further testing.

Learning by doing virtually.

Selective reduction of bone without collateral damage (nerves, teeth) is essential in apicectomy. To test whether skills acquired on a virtual apicectomy simulator (VOXEL-MAN system with integrated force-feedback) are transferable from virtual to physical reality, two groups of trainees were compared. Group 1 received computer-based virtual surgical training before performing an apicectomy in a pig cadaver model. The probability of preserving vital neighboring structures was improved significantly, i.e. six-fold, after virtual surgical training (P<0.001). The average volume of the bony defects created by the trainees of Group 2 (mean: 0.47 ml) was significantly (P<0.001) larger than by the trainees of Group 1 (mean: 0.25 ml). Most importantly, the ability to objectively self-assess performance was significantly improved after virtual training. Training with a virtual apicectomy simulator appears to be effective, and the skills acquired are transferable to physical reality.

Construction of a PAC vector system for the propagation of genomic DNA in bacterial and mammalian cells and subsequent generation of nested deletions in individual library members.

The BAC and PAC cloning systems allow investigators to propagate large genomic DNA fragments up to 300 kb in size in E. colicells. We describe a new PAC shuttle vector that can be propagated in both bacterial and human cells. Specifically, the P1 cloning vector pAd10sacBII was modified by the insertion of a puromycin-resistance gene (pac), the Epstein-Barr Virus (EBV) latent replication origin oriP,and the EBV EBNA1 gene. Transfection studies in HEK 293 cells demonstrated that the modified vector was stably maintained as an episome for at least 30 generations. And since pJCPAC-Mam1 contains a loxP site, genomic DNA cloned into this vector can be subjected to loxP-Cre -mediated deletion events. The transposon vector pTnPGKpuro/loxP was modified to make this system amenable to propagation in human cells by inserting pac, oriP, and EBNA1 elements into the vector (Chatterjee, P.K., Coren, J.C., 1997. Isolating large nested deletions in PACs and BACs by in vivo selection of P1 headful-packaged products of Cre-catalyzed recombination between the loxP site in PAC and BAC and one introduced in transposition. NAR 25, 2205-2212.). pTnPGKpuro/loxP-EBV was then used to generate deletions in an individual library member to demonstrate that all of the deletions still contain the required eukaryotic elements and that they were nested. All library members constructed in pJCPAC-Mam1 can be directly transformed into human cells to assess function. And the deletion technology can be used to aid in delineating the boundaries of genes and other cis-acting elements.

Construction of bacteriophage P1 libraries with large inserts.

The bacteriophage P1 cloning system was originally developed as an alternative to YAC and cosmid systems for cloning high-molecular-weight genomic DNA. This unit details the preparation of the bacteriophage P1 library. Three support protocols provide the raw materials for the basic procedure, including the vector (pAd10sacBII), the mammalian DNA inserts, and the two packaging extracts that contain the viral proteins necessary to construct a P1 bacteriophage incorporating the vector and insert. A fourth support protocol describes how to induce replication of the plasmids cloned in the basic protocol, isolate the cloned DNA, and analyze the final products.

Screening large-insert libraries by PCR.

This unit describes procedures for screening large-insert libraries by multistep polymerase chain reaction (PCR) analysis of DNA samples from clone pools. In the basic protocol, PCR amplification and agarose gel electrophoresis are used to identify successively smaller pools of DNA or colonies that contain clones with the appropriate-sized amplification product. In the last screening step, individual clones are identified. The first and second support protocols describe the preparation of DNA and yeast-cell pools for screening, and the first alternate protocol describes the preparation of crude lysates suitable for PCR from individual clones or small-scale pools.

The bacteriophage P1 lytic replicon: directionality of replication and cis-acting elements.

We have identified the direction of replication of a bacteriophage P1 lytic replicon. This was accomplished by constructing lambda P1 lysogens that contain a functional P1 lytic replicon and analysing which of two nearby bacterial DNA markers flanking the lambda prophage were amplified when that replicon was activated. We demonstrate that both DNA markers are coordinately amplified, a result consistent with lytic replication proceeding in a bidirectional fashion. To analyze the role of various elements comprising the lytic replicon, we assessed the ability of a wild type replicon to complement a defective replicon that contains a transposon inserted between an essential lytic replication gene (repL) and the promoter (P53) at which transcription of that gene is initiated. We show that the wild type replicon cannot complement the mutant replicon. The simplest hypothesis to explain this result is that either P53 or repL protein functions primarily in cis for the replicon to operate.

Retrofitting high molecular weight DNA cloned in P1: introduction of reporter genes, markers selectable in mammalian cells and generation of nested deletions.

The bacteriophage P.1. cloning system is proving to be quite useful for the cloning and analysis of genomic DNA inserts of up to 95 kb in size. In an effort to use that DNA directly in biological experiments we have embarked on a scheme to retrofit the P.1. DNA using a mini-Tn10 transposon system. This transposon system is used in two ways: (i) to introduce a variety of sequence signals that are recognizable in mammalian cells, such as mammalian cell-responsive resistance markers and reporter genes, and (ii) to generate a nested set of deletions in a P.1. clone by using a ioxP site located within the transposon. In this report we show that such transpositions into P.1. DNA are efficient, distributed throughout the entire length of the genomic fragment and do not disrupt the DNA in any location other than the site of insertion of the transposon. The Tn10-based P.1. transduction system described here provides a general scheme for retrofitting any large genomic DNA cloned in a P.1. vector, thus facilitating the use of clones from the current P.1. recombinant libraries in cellular transformation studies.

A general genetic approach in Escherichia coli for determining the mechanism(s) of action of tumoricidal agents: application to DMP 840, a tumoricidal agent.

We describe here a simple and easily manipulatable Escherichia coli-based genetic system that permits us to identify bacterial gene products that modulate the sensitivity of bacteria to tumoricidal agents, such as DMP 840, a bisnaphthalimide drug. To the extent that the action of these agents is conserved, these studies may expand our understanding agents is conserved, these studies may expand our understanding of how the agents work in mammalian cells. The approach briefly is to use a library of E. coli genes that are overexpressed in a high copy number vector to select bacterial clones that are resistant to the cytotoxic effects of drugs. AtolC bacterial mutant is used to maximize permeability of cells to hydrophobic organic molecules. By using DMP 840 to model the system, we have identified two genes, designated mdaA and mdaB, that impart resistance to DMP 840 when they are expressed at elevated levels. mdaB maps to E. coli map coordinate 66, is located between the parE and parC genes, and encodes a protein of 22 kDa. mdaA maps to E. coli map coordinate 18, is located adjacent to the glutaredoxin (grx) gene, and encodes a protein of 24 kDa. Specific and regulatable overproduction of both of these proteins correlates with DMP 840 resistance. Overproduction of the MdaB protein also imparts resistance to two mammalian topoisomerase inhibitors, Adriamycin and etoposide. In contrast, overproduction of the MdaA protein produces resistance only to Adriamycin. Based on its drug-resistance properties and its location between genes that encode the two subunits of the bacterial topoisomerase IV, we suggest that mdaB acts by modulating topoisomerase IV activity. The location of the mdaA gene adjacent to grx suggests it acts by a drug detoxification mechanism.

Headful packaging revisited: the packaging of more than one DNA molecule into a bacteriophage P1 head.

Like a variety of other bacteriophages, such as T4 and P22, bacteriophage P1 packages DNA by a "headful" mechanism in which the capacity of the viral capsid determines the size of the single DNA molecule that is packaged. Because of the long-standing and general acceptance of this packaging mechanism, we were surprised to discover that some of our observations, using the in vitro P1 packaging system, could be explained by the packaging of less than headful-sized (< 110 kb) DNA molecules into a P1 capsid. To account for these observations, we describe results that support a model of in vitro P1 packaging in which multiple less than headful-sized DNA molecules are taken into a P1 head until that head has been filled. The results further suggest that the phage so generated can occasionally inject more than one DNA molecule into a cell upon viral infection. The data that supports these conclusions are: (1) the DNAs of the circular P1 cloning vectors pAd10sacBII (32 kb) and pNS358 (14 kb) are packaged in vitro with an efficiency of about 6 to 12% of that of longer concatemers of these DNAs. (2) The in vitro packaging of two differentially marked, less than 18 kb plasmid DNAs in the same reaction results in the production of a phage that can occasionally inject both DNAs into the same cell upon infection. (3) Virus particles generated by the packaging of either pAd10sacBII plasmid DNA or the two differently marked plasmids have a density in CsCl equilibrium gradients that is the same as P1 plaque-forming phage, suggesting that the former phage contain a headful of DNA. These results cannot be explained by Cre-mediated site-specific recombination between plasmids in the P1 packaging extracts. Finally, we present in vivo experiments that are also consistent with the headful packaging of multiple DNAs into a P1 head.

Using cell-fractionation and photochemical crosslinking methods to determine the cellular binding site(s) of the antitumor drug DMP 840.

In order to understand its mechanism of action we have begun an effort to better define the cellular target of action of the experimental antitumor agent DMP 840 (NSC D640430; (R,R)-2,2'-(1,2-ethanediylbis(imino-(1-methyl-2,1-ethanediyl)))-bi s(5- nitro-1H-benz(de)isoquinoline-1,3-(2H)-dione) dimethanesulfonate). Using a combination of gentle cell fractionation procedures and a previously unidentified photochemical crosslinking reaction, we have shown that after the drug is added to cultured Clone A cells, more than 80% of the drug that is found associated with cells partitions to the chromatin-containing structural framework of the cell and that the primary target after crosslinking with 360 nm light is DNA. While DMP 840 photoreacts quite efficiently with purified RNA in vitro, no photoattachment of the drug to RNA was observed in cells. In vitro photochemical studies also reveal that while GC-rich DNA is a preferred target for drug interaction, AT-rich DNA is more active in the photochemical crosslinking reaction. These results suggest that DMP 840 probably kills cells by interfering with DNA-metabolic processes, and that the drug and its derivatives are likely to be useful photoactive molecular probes for investigating higher order chromatin structures in cells.

Display of peptides and proteins on the surface of bacteriophage lambda.

The display of peptides or proteins on the surface of viruses is an important technology for studying peptides or proteins and their interaction with other molecules. Here we describe a display vehicle based on bacteriophage lambda that incorporates a number of features distinct from other currently used display systems. Fusions of peptides or protein domains have been made to the amino terminus of the 11-kDa D protein of the lambda capsid. These fusions assemble onto the viral capsid and appear to be accessible to ligand interactions, based on the ability of a monoclonal antibody to recognize an epitope fused to the D protein on phage heads. To produce large D fusion display libraries and yet avoid the cumbersome task of cloning many fragments into lambda DNA, we have used the Cre-loxP site-specific recombination system in vivo to incorporate plasmids encoding the D fusions into the phage genome. Finally, we show that D fusion proteins can be added in vitro to phage lacking D protein and be assembled onto the viral capsid.

Purification and DNA-binding activity of the PacA subunit of the bacteriophage P1 pacase enzyme.

The bacteriophage P1 packaging site (pac) cleavage enzyme (pacase) consists of two phage encoded proteins, PacA and PacB. Both proteins are necessary for the recognition and cleavage of pac and for subsequent packaging of cleaved DNA into phage particles. We have purified PacA to homogeneity from a bacterial strain that overproduces the protein. Purified PacA complements an Escherichia coli extract containing the PacB protein for DNA cleavage at the pac site and recognizes and binds to methylated pac DNA independently of PacB in gel retardation experiments. The latter property of PacA is absolutely dependent on the presence of a wildtype E. coli extract, suggesting that E. coli host proteins play a role in the pac cleavage reaction.

Faithful cleavage of the P1 packaging site (pac) requires two phage proteins, PacA and PacB, and two Escherichia coli proteins, IHF and HU.

The PacA and PacB subunits of the bacteriophage P1 DNA packaging enzyme (pacase) are necessary for cleavage of the phage packaging site (pac). In the accompanying paper, we show that the PacA subunit of the enzyme specifically binds to pac in the absence of PacB, but requires factors present in an Escherichia coli extract to do so. We show here that either of two E. coli DNA binding proteins, integration host factor (IHF) or HU, can replace this extract and promote the binding of PacA to pac. IHF binds to pac independently of PacA and DNase I footprinting experiments show that IHF protects approximately 40 bp of DNA around an IHF consensus sequence adjacent to the cleavage site. DNase I footprinting experiments also show that in the presence of either IHF or HU, PacA binds to the hexanucleotide sequences (5'-TGATCA/G) that flank the cleavage site and that have been previously shown to be essential for pac cleavage. The importance of IHF and HU in pac cleavage is further demonstrated by the severe reduction in both the fidelity and efficiency of pac cleavage in vitro with extracts lacking both IHF and HU. Addition of either IHF or HU to the deficient extracts renders them fully proficient for pac cleavage. Finally, we show that IHF bends DNA at the IHF site within pac. Based on these results, we propose a model that can account for the role of the various phage and host proteins, and for DNA bending in the pac cleavage reaction.

Preparation and screening of an arrayed human genomic library generated with the P1 cloning system.

We describe here the construction and initial characterization of a 3-fold coverage genomic library of the human haploid genome that was prepared using the bacteriophage P1 cloning system. The cloned DNA inserts were produced by size fractionation of a Sau3AI partial digest of high molecular weight genomic DNA isolated from primary cells of human foreskin fibroblasts. The inserts were cloned into the pAd10sacBII vector and packaged in vitro into P1 phage. These were used to generate recombinant bacterial clones, each of which was picked robotically from an agar plate into a well of a 96-well microtiter dish, grown overnight, and stored at -70 degrees C. The resulting library, designated DMPC-HFF#1 series A, consists of approximately 130,000-140,000 recombinant clones that were stored in 1500 microtiter dishes. To screen the library, clones were combined in a pooling strategy and specific loci were identified by PCR analysis. On average, the library contains two or three different clones for each locus screened. To date we have identified a total of 17 clones containing the hypoxanthine-guanine phosphoribosyltransferase, human serum albumin-human alpha-fetoprotein, p53, cyclooxygenase I, human apurinic endonuclease, beta-polymerase, and DNA ligase I genes. The cloned inserts average 80 kb in size and range from 70 to 95 kb, with one 49-kb insert and one 62-kb insert.

Three new developments in P1 cloning. Increased cloning efficiency, improved clone recovery, and a new P1 mouse library.

In this report, we describe three new P1 cloning developments. Two of these developments represent improvements in cloning efficiency and clone recovery, and the third is the production and partial characterization of a new P1 mouse library. To increase cloning efficiency, we have produced a new lysis-defective (delta lydAB) P1 lysogen (NS3690) for the production of the stage II head-tail-P1 packaging extract that is easier to use than the original stage II lysogen (NS3210), and that produces stage II extracts that are five- to eightfold more efficient than the original extracts. We believe the increased efficiency is due to the more concentrated packaging components in the NS3690 extract. Regarding P1 clone recovery, we demonstrate here that the less than optimal recovery of P1 plasmid DNA from P1 clones is due to the continuous presence of the P1 Cre recombinase in the host strain containing those clones (NS3529). Consequently, a simple method of P1 plasmid clone transduction is described to transfer clone DNA from NS3529 (Cre+) to its Cre- parent (NS3516). Yields of P1 plasmid DNA from NS3516 are as much as tenfold higher than from NS3529. Finally, we document here the production of a new P1 mouse library that was generated using genomic DNA from embryonic stem cell line E14 (a 129/0la mouse). The library contains 182,000 independent clones whose average insert size is 80 kb and, based on > 100 polymerase chain reaction screens, has an average unique sequence-hit size of 4.6.

P1 and cosmid clones define the organization of 280 kb of the mouse H-2 complex containing the Cps-1 and Hsp70 loci.

A 280 kilobase (kb) contig was isolated from mouse genomic P1 and cosmid libraries, using as probes human cDNA and genomic DNA fragments that map in the interval between the second component of complement and tumor necrosis factor genes of the HLA complex. The clone contig demonstrates synteny of eleven mouse genes that are homologous to genes initially mapped within the human major histocompatibility complex. These include the mouse homologs of BAT2 (HLA-B-associated transcript 2) through BAT9 and also three HSP70-related genes. Five P1 clones form a contig of 240 kb that spans from BAT9 through BAT3. Twelve cosmid clones are arranged in three contigs that confirm most of the structure of the P1 contig and link the mouse BAT3 homolog to the BAT2 homolog approximately 15 kb farther telomeric. Polymorphic DNA markers within the cloned region were used to map the cleft palate susceptibility-1 (Cps-1) locus to the interval between Hsp70.1 and BAT6 (valyl-tRNA synthetase). This refines the location of the Cps-1 locus to a 45 kb region contained in the H2-124 P1 insert.

A positive selection vector for cloning high molecular weight DNA by the bacteriophage P1 system: improved cloning efficacy.

The bacteriophage P1 cloning system can package and propagate DNA inserts that are up to 95 kilobases. Clones are maintained in Escherichia coli by a low-copy replicon in the P1 cloning vector and can be amplified by inducing a second replicon in the vector with isopropyl beta-D-thiogalactopyranoside. To overcome the necessity of screening clones for DNA inserts, we have developed a P1 vector with a positive selection system that is based on the properties of the sacB gene from Bacillus amyloliquefaciens. Expression of that gene kills E. coli cells that are grown in the presence of sucrose. In the new P1 vector (pAd10sacBII) sacB expression is regulated by a synthetic E. coli promoter that also contains a P1 C1 repressor binding site. A unique BamHI cloning site is located between the promoter and the sacB structural gene. Cloning DNA fragments into the BamHI site interrupts sacB expression and permits growth of plasmid-containing cells in the presence of sucrose. We have also bordered the BamHI site with unique rare-cutting restriction sites Not I, Sal I, and Sfi I and with T7 and Sp6 promoter sequences to facilitate characterization and analysis of P1 clones. We describe here the use of Not I digestion to size the cloned DNA fragments and RNA probes to identify the ends of those fragments. The positive selection P1 vector provides a 65- to 75-fold discrimination of P1 clones that contain inserts from those that do not. It therefore permits generation of genomic libraries that are much easier to use for gene isolation and genome mapping than are our previous libraries. Also, the new vector makes it feasible to generate P1 libraries from small amounts of genomic insert DNA, such as from sorted chromosomes.

Bacteriophage P1 genes involved in the recognition and cleavage of the phage packaging site (pac).

The packaging of bacteriophage P1 DNA is initiated by cleavage of the viral DNA at a specific site, designated pac. The proteins necessary for that cleavage, and the genes that encode those proteins, are described in this report. By sequencing wild-type P1 DNA and DNA derived from various P1 amber mutants that are deficient in pac cleavage, two distinct genes, referred to as pacA and pacB, were identified. These genes appear to be coordinately transcribed with an upstream P1 gene that encodes a regulator of late P1 gene expression (gene 10). pacA is located upstream from pacB and contains the 161 base-pair pac cleavage site. The predicted sizes of the PacA and PacB proteins are 45 kDa and 56 kDa, respectively. These proteins have been identified on SDS-polyacrylamide gels using extracts derived from Escherichia coli cells that express these genes under the control of a bacteriophage T7 promoter. Extracts prepared from cells expressing both PacA and PacB are proficient for site-specific cleavage of the P1 packaging site, whereas those lacking either protein are not. However, the two defective extracts can complement each other to restore functional pac cleavage activity. Thus, PacA and PacB are two essential bacteriophage proteins required for recognition and cleavage of the P1 packaging site. PacB extracts also contain a second P1 protein that is encoded within the pacB gene. We have identified this protein on SDS-polyacrylamide gels and have shown that it is translated in the same reading frame as is PacB. Its role, if any, in pac cleavage is yet to be determined.