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.

Cloning high molecular weight DNA fragments by the bacteriophage P1 system.

The cloning of high molecular weight genomic DNA promises to provide the means of mapping chromosomes, isolating genes, and understanding long-range effects on gene expression. This review describes the background and some recent advances in cloning of high molecular weight DNA using the bacteriophage P1 system.

Model for homologous recombination during transfer of DNA into mouse L cells: role for DNA ends in the recombination process.

We have constructed phage lambda and plasmid DNA substrates (lambda tk2 and ptk2) that contain two defective herpesvirus thymidine kinase (tk) genes that can be used to detect homologous recombination during the transfer of DNA into mouse L cells deficient in thymidine kinase activity. The recombination event reconstructs a wild-type tk gene and is scored because it converts Tk- cells to Tk+. Using this system, we have shown that (i) both intramolecular and intermolecular homologous recombination can be detected after gene transfer; (ii) the degree of recombination decreases with decreasing tk gene homology; and (iii) the efficiency of recombination can be stimulated 10- to 100-fold by cutting the tk2 DNA with restriction enzymes at appropriate sites relative to the recombining sequences. Based on the substrate requirements for these recombination events, we propose a model to explain how recombination might occur in mammalian cells. The essential features of the model are that the cut restriction site ends are substrates for cellular exonucleases that degrade DNA strands. This process exposes complementary strands of the two defective tk genes, which then pair. Removal of unpaired DNA at the junction between the paired and unpaired regions permits a gap repair process to reconstruct an intact gene.

Homologous recombination between overlapping thymidine kinase gene fragments stably inserted into a mouse cell genome.

We have constructed a substrate to study homologous recombination between adjacent segments of chromosomal DNA. This substrate, designated lambda tk2 , consists of one completely defective and one partially defective herpes simplex virus thymidine kinase (tk) gene cloned in bacteriophage lambda DNA. The two genes have homologous 984-base-pair sequences and are separated by 3 kilobases of largely vector DNA. When lambda tk2 DNA was transferred into mouse LMtk- cells by the calcium phosphate method, rare TK+ transformants were obtained that contained many (greater than 40) copies of the unrecombined DNA. Tk- revertants, which had lost most of the copies of unrecombined DNA, were isolated from these TK+-transformed lines. Two of these Tk- lines were further studied by analysis of their reversion back to the Tk+ phenotype. They generated ca. 200 Tk+ revertants per 10(8) cells after growth in nonselecting medium for 5 days. All of these Tk+ revertants have an intact tk gene reconstructed by homologous recombination; they also retain various amounts of unrecombined lambda tk2 DNA. Southern blot analysis suggested that at least some of the recombination events involve unequal sister chromatid exchanges. We also tested three agents, mitomycin C, 12-O-tetradecanoyl-phorbol-13-acetate, and mezerein, that are thought to stimulate recombination to determine whether they affect the reversion from Tk- to Tk+. Only mitomycin C increased the number of Tk+ revertants.

Intermolecular recombination between DNAs introduced into mouse L cells is mediated by a nonconservative pathway that leads to crossover products.

We describe experiments designed to measure the efficiency of intermolecular recombination between mutant herpesvirus thymidine kinase (tk) genes introduced into mouse L cells. Recombinants were scored as stable transformants containing a functional tk gene. The two recombination substrates used were ptkB8, a pBR322-based plasmid containing a mutant tk gene, with a BamHI linker in an SphI restriction site that is centrally located within the gene, and mp10tk delta 3' delta 5', an mp10 vector with a tk gene deleted at both the 3' and 5' ends. The only homology shared by the two DNAs is 885 base pairs within the tk gene. To determine whether the double-strand break repair model that has been used to explain recombination in yeast cells (J. W. Szostak, T. L. Orr-Weaver, R. J. Rothstein, and F. W. Stahl, Cell 33:25-35, 1983) can account for recombination during the introduction of these DNAs into mammalian cells, we transformed cells with BamHI-linearized ptkB8 and supercoiled mp10tk delta 3' delta 5' replicative-form DNA. These two DNAs should recombine efficiently according to that model and should generate gene conversion products. In this reaction, the supercoiled DNA acts as the donor of information to repair the cleaved tk gene. Our results indicated that the efficiency of this reaction was very low (less than 10 transformants were obtained per 0.1 microgram of each DNA used in the reaction per 10(6) cells). In contrast, if BamHI-cleaved ptkB8 DNA was cotransformed into cells along with a circular DNA molecule containing a tk gene deleted only at its 3' end or only at its 5' end (mp10tk delta 3' or mp10tk delta 5'), then the efficiency of recombination could be more than 4 orders of magnitude higher than it was with circular mp10tk delta 3' delta 5' DNA. Recombination frequencies were highest when the tk delta 3' or tk delta 5' DNA used was cleaved at the tk deletion junction. Southern analyses of DNA from TK+ transformants generated with BamHI-cleaved ptkB8 and BamHI-cleaved mp10tk delta 3' DNAs indicated that recombination was almost always associated with the reassortment of markers flanking the reconstructed tk DNA. Together, these results are more consistent with the nonconservative single-strand annealing model for recombination that we proposed several years ago (F.-L. Lin, K. Sperle, and N. Sternberg, Mol. Cell. Biol. 4:1020-1034, 1984) than they are with the double-strand break repair model.

Repair of double-stranded DNA breaks by homologous DNA fragments during transfer of DNA into mouse L cells.

To test the validity of various models for recombination between extrachromosomal DNAs in mammalian cells, we measured recombination between a plasmid containing a herpesvirus thymidine kinase (tk) gene with an internal BamHI linker insertion mutation (ptkB8) and a tk gene deleted at both ends (tk delta 3' delta 5'). The two DNAs shared 885 base pairs of perfect tk homology except for the interruption at the linker insertion site. Recombination events that restored the mutated insertion site to wild type were monitored by the generation of hypoxanthine-aminopterine-thymidine-resistant colonies after cotransformation of Ltk- cells with the two DNAs. We found that cleavage of the ptkB8 DNA at the linker insertion site was essential for gene restoration. If the tk delta 3' delta 5' DNA was ligated into mp10 vector DNA, then recombination with the cleaved ptkB8 DNA was inefficient. In contrast, if it was excised from that vector by cleavage at flanking restriction sites, then recombination was stimulated about 150-fold. Using restriction site polymorphisms, we showed that most of the recombination events leading to restoration of the tk gene with the excised tk delta 3' delta 5' fragment involved three double-strand duplexes: two ptkB8 DNAs and one tk delta 3' delta 5' fragment. These results are much more readily explained by the single-strand annealing model of recombination than by the double-strand break repair model, and they suggest that the deficiency of the latter pathway for extrachromosomal mammalian recombination may be due, at least in part, to the obligate tripartite nature of the reaction. Finally, we measured the effect of DNA homology on the efficiency of the ptkB8-tk delta 3' delta 5' reaction. Our results showed a near-linear relationship between the efficiency of recombination and the amount of homology flanking either side of the linker insertion site. Moreover, we could detect thymidine kinase-positive transformants with as little as 10 base pairs of homology.

Extrachromosomal recombination in mammalian cells as studied with single- and double-stranded DNA substrates.

We have previously proposed a model to account for the high levels of homologous recombination that can occur during the introduction of DNA into mammalian cells (F.-L. Lin, K. Sperle, and N. Sternberg, Mol. Cell. Biol. 4:1020-1034, 1984). An essential feature of that model is that linear molecules with ends appropriately located between homologous DNA segments are efficient substrates for an exonuclease that acts in a 5'----3' direction. That process generates complementary single strands that pair in homologous regions to produce an intermediate that is processed efficiently to a recombinant molecule. An alternative model, in which strand degradation occurs in the 3'----5' direction, is also possible. In this report, we describe experiments that tested several of the essential features of the model. We first confirmed and extended our previous results with double-stranded DNA substrates containing truncated herpesvirus thymidine kinase (tk) genes (tk delta 5' and tk delta 3'). The results illustrate the importance of the location of double-strand breaks in the successful reconstruction of the tk gene by recombination. We next transformed cells with pairs of single-stranded DNAs containing truncated tk genes which should anneal in cells to generate the recombination intermediates predicted by the two alternative models. One of the intermediates would be the favored substrate in our original 5'----3' degradative model and the other would be the favored substrate in the alternative 3'----5' degradative model. Our results indicate that the intermediate favored by the 3'----5' model is 10 to 20 times more efficient in generating recombinant tk genes than is the other intermediate.

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.

Recognition and cleavage of the bacteriophage P1 packaging site (pac). II. Functional limits of pac and location of pac cleavage termini.

Bacteriophage P1 initiates the processive packaging of its DNA at a unique site called pac. We show that a functional pac site is contained within a 161 base-pair segment of P1 EcoRI fragment 20. It extends from a position 71 base-pairs to a position 232 base-pairs from the EcoRI-22 proximal side of that fragment. The 3' and 5' pac termini are located centrally within that 161 base-pair region and are distributed over about a turn of the DNA helix. The DNA sequence of the terminus region is shown below, with the large arrows indicating the positions of termini that are frequently represented in the PI population and the small arrows indicating the positions of termini that are rarely represented in the P1 population. (Sequence: in text). Digestion of P1 virus DNA with EcoRI generates two major EcoRI-pac fragments, which differ in size by about five or six base-pairs. While the structure and position of the double-stranded pac ends of these fragments have not been determined precisely, the 5' termini at those ends probably correspond to the two major pac cleavage sites in the upper strand of the sequences shown above. The 161 base-pair pac site contains the hexanucleotide sequence 5'-TGATCAG-3' repeated four times at one end and three times at the other. Removal of just one of those elements from either the right or left ends of pac reduces pac cleavage by about tenfold. Moreover, the elements appear to be additive in their effect on pac cleavage, as removal of one and a half elements or all three elements from the right side of pac reduces pac cleavage 100-fold, and greater than 1000-fold, respectively.

Recognition and cleavage of the bacteriophage P1 packaging site (pac). I. Differential processing of the cleaved ends in vivo.

The packaging of bacteriophage P1 DNA into viral capsids is initiated at a specific DNA site called pac. During packaging, that site is cleaved and at least one of the resulting ends is encapsidated into a P1 virion. We show here that pac is located on a 620 base-pair fragment of P1 DNA (EcoRI-20). When that fragment is inserted into the chromosome of cells that are then infected with P1, packaging of host DNA into phage particles is initiated at pac and proceeds down the chromosome, unidirectionally, for about five to ten P1 "headfuls" (about 5 X 10(5) to 10 X 10(5) bases of DNA). Using an assay for pac cleavage that does not depend on DNA packaging, we have identified a set of five amber mutations that are mapped adjacent to pac, and that define a gene (gene 9) essential for pac cleavage. Amber mutations that are located in genes necessary for viral capsid formation (genes 4, 8 and 23), or in a gene necessary for "late" protein synthesis (gene 10), do not affect pac cleavage. The latter result suggests that the synthesis of the pac cleavage protein is not regulated co-ordinately with other phage morphogenesis proteins. The products of pac cleavage were analyzed using two different DNA substrates. In one case, a single copy of pac was placed in the chromosome of P1-sensitive cells. When those cells were infected with P1, we could detect the cleavage of as much as 70% of the pac-containing DNA. The pac end destined to be packaged in the virion was detected five to 20 times more efficiently than was the other end. Since this result is obtained whether or not the infecting P1 phage can encapsidate the cut pac site, the differential detection of pac ends is not simply a consequence of one end being packaged and the other not. In a second case, pac was located in cells on a small (5 X 10(3) bases) multicopy plasmid. When those cells were infected with P1, neither pac end was detected efficiently after P1 infection, unless the cells carried a recBCD- mutation. In recBCD- cells, the results with plasmid-pac substrates were similar to those obtained with chromosomally integrated pac substrates. We interpret these results to mean that, following pac cleavage, the end destined to be packaged is protected from cellular nucleases while the other end is degraded by the action of at least two nucleases, one of which is the product of the host recBCD gene.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetic analysis of the lytic replicon of bacteriophage P1. I. Isolation and partial characterization.

Despite the extensive genetic analysis of bacteriophage P1, the region of the viral genome that is responsible for its lytic (vegetative) replication has not been identified. In this paper we describe the identification of various fragments of P1 DNA that can replicate an otherwise replication-defective lambda vector when they are cloned into that vector. The fragments share a 2800 base-pair segment of the P1 genome that is located adjacent to the immI region of the phage. Replication mediated by the cloned P1 fragments is abolished by the product of the P1 c1 gene, the repressor of phage lytic functions. Since these properties resemble those of the P1 lytic replicon, we suggest that the 2800 base-pair segment identified here contains that replicon.

Genetic analysis of the lytic replicon of bacteriophage P1. II. Organization of replicon elements.

The region of bacteriophage P1 DNA containing a lytic (vegetative) replicon has been identified by cloning P1 fragments into a phage lambda vector. We present the sequence of that replicon. Using a novel fusion vector containing two P1 loxP recombination sites, we have developed a transformation assay for replicon function and have used that assay to identify some of the components of the P1 lytic replicon. Among those components is a transcription promoter, P53, whose activity is essential for replicon function. When that promoter is inactivated by the binding of P1 repressor to an operator site, Op53, whose sequence overlaps the promoter, replicon function is blocked. The P53 promoter can be replaced for replicon function by other promoters and, when the lacZ promoter was used, the extent of replication was shown to be proportional to promoter activity. Two open reading frames are located downstream from P53. The promoter-proximal reading frame is 266 amino acid residues long and is not essential for replicon function. In fact, expression of that open reading frame either interferes with plasmid establishment after transformation or is lethal to cells. The promoter-distal reading frame, designated the repL open reading frame, is either 269 or 281 amino acid residues long and is essential for replicon function. Insertion of a Tn5 transposon into the 266 amino acid residue open reading frame inactivates the cloned lytic replicon probably by interfering with the transcription of the repL open reading frame from P53. In P1, this Tn5 insertion mutation completely blocks lytic replication, indicating that the replicon identified here is either the only P1 lytic replicon or, if not, is at least necessary for the function of any other lytic replicon. A four base insertion in the repL open reading frame has largely the same inhibitory effect on phage lytic replication as the Tn5 insertion.

Characterization of the binding sites of c1 repressor of bacteriophage P1. Evidence for multiple asymmetric sites.

The repressor of bacteriophage P1, encoded by the c1 gene, is responsible for maintaining a P1 prophage in the lysogenic state. In this paper we present: (1) the sequence of the rightmost 943 base-pairs of the P1 genetic map that includes the 5'-terminal 224 base-pairs of the c1 gene plus its upstream region; (2) the construction of a plasmid that directs the production of approximately 5% of the cell's protein as P1 repressor; (3) a deletion analysis that establishes the startpoint of P1 repressor translation; (4) filter binding experiments that demonstrate that P1 repressor binds to several regions upstream from the c1 gene; (5) DNase I footprint experiments that directly identify two of the P1 repressor binding sites. Sequences very similar to the identified binding sites occur in at least 11 sites in P1, in most cases near functions known, or likely, to be controlled by repressor. From these sites we have derived the consensus binding site sequence ATTGCTCTAATAAATTT. We suggest that, unlike other phage operators, the P1 repressor binding sites lack rotational symmetry.

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.

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.

Bacteriophage P1 cre gene and its regulatory region. Evidence for multiple promoters and for regulation by DNA methylation.

The bacteriophage P1 site-specific recombination system consists of two components, a site, loxP, at which recombination occurs, and a recombinase protein, Cre. In this paper, we present the DNA sequence of the cre structural gene and its upstream regulatory region. Analysis of the sequence indicates: (1) that cre encodes a protein of 343 amino acids; (2) that cre and loxP are separated by a 434 base-pair region that contains a 73 amino acid open reading frame, orf1; and (3) that cre and orf1 are oriented with their amino-terminal ends proximal to loxP. We have identified three promoters that are located upstream of the cre structural gene. Their activities range from 7 to 10% of the activity of the galactose operon promoter. The promoter furthest from cre, pR1, contains two Dam methylation sites (5'-G-A-T-C-3') in its -35 region, and is sensitive to Dam methylation. Its transcription is three- to fourfold higher in a dam- host than it is in a dam+ host. The promoter closest to cre, pR3, signals the production of an RNA transcript that functions inefficiently for Cre protein synthesis because it lacks a ribosome recognition site. None of the three cre promoters is sensitive to proteins expressed by the P1 prophage, including the c1 repressor protein. To assess the role of cre in the P1 life-cycle, we isolated cre mutants and studied their behavior in recA+ and recA- hosts. Those studies indicate that Cre is dispensable for viral vegetative growth and lysogeny in a recA+ host, but is required for both processes in a recA- host. The cre requirement for lysogeny suggests that the protein is essential for the cyclization of newly injected terminally redundant virion DNA. The requirement for vegetative growth suggests that Cre also has a role to play in the viral lytic cycle after the viral DNA has been cyclized.

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.

The production of generalized transducing phage by bacteriophage lambda.

Generalized transduction has for about 30 years been a major tool in the genetic manipulation of bacterial chromosomes. However, throughout that time little progress has been made in understanding how generalized transducing particles are produced. The experiments presented in this paper use phage lambda to assess some of the factors that affect that process. The results of those experiments indicate: the production of generalized transducing particles by bacteriophage lambda is inhibited by the phage lambda exonuclease (Exo). Also inhibited by lambda Exo is the production of lambda docR particles, a class of particles whose packaging is initiated in bacterial DNA and terminated at the normal phage packaging site, cos. In contrast, the production of lambda docL particles, a class of particles whose packaging is initiated at cos and terminated in bacterial DNA, is unaffected by lambda Exo; lambda-generalized transducing particles are not detected in induced lysis-defective (S-) lambda lysogens until about 60-90 min after prophage induction. Since wild-type lambda would normally lyse cells by 60 min, the production of lambda-generalized transducing particles depends on the phage being lysis-defective; if transducing lysates are prepared by phage infection then the frequency of generalized transduction for different bacterial markers varies over a 10-20-fold range. In contrast, if transducing lysates are prepared by the induction of a lambda lysogen containing an excision-defective prophage, then the variation in transduction frequency is much greater, and markers adjacent to, and on both sides of, the prophage are transduced with much higher frequencies than are other markers; if the prophage is replication-defective then the increased transduction of prophage-proximal markers is eliminated; measurements of total DNA in induced lysogens indicate that part of the increase in transduction frequency following prophage induction can be accounted for by an increase in the amount of prophage-proximal bacterial DNA in the cell. Measurements of DNA in transducing particles indicate that the rest of the increase is probably due to the preferential packaging of the prophage-proximal bacterial DNA. These results are most easily interpreted in terms of a model for the initiation of bacterial DNA packaging by lambda, in which the proteins involved (Ter) do not recognize any particular sequence in bacterial DNA but rather recognize some feature of the DNA tht is sensitive to lambda exonuclease, such as a nick or a double-stranded cut.(ABSTRACT TRUNCATED AT 400 WORDS)

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.