Physical characterization of bacteriophage MX4, a generalized transducing phage for Myxococcus xanthus.

A generalized transducing bacteriophage of Myxococcus xanthus has been examined. The phage particle consists of an isometric head and a contractile tail. The genome of the phage is a linear DNA molecule of molecular weight 39 ± 2.1 × 10⁶, which contains the normal DNA bases 70% of which are guanosine + cytosine. No overall heterogeneity of base composition is present. The DNA does not carry easily detectable cohesive ends nor is it cyclically permuted. It does contain a large and somewhat variable terminal redundancy. Heating phage particles in the presence of EDTA causes tail sheath contraction and ejection of DNA, some of which remains attached to the tail. Digestion of tail-bound DNA with restriction enzymes shows that the phage tail can be attached to either end of the DNA. Thus the DNA probably contains recognition sites for the packaging of its DNA at both ends. These results suggest possible mechanisms for the genesis of transducing particles by phage MX4.     

The action of mutagens on MS2 phage and on its infective RNA. V. Kinetics of the chemical and functional changes of the genome under hydroxylamine treatment

The kinetics of chemical and functional changes induced in the genome of bacteriophage MS2 by hydroxylamine under the conditions of predominant modification of either cytidine (pH 5.0) or uridine (pH 8.0) have been studied.Comparison of the kinetics of chemical modifications of monomeric nucleotides with those of bacteriophage inactivation at pH 8.0 and 5.0 made it possible to estimate the effective number of exposed cytidine and uridine residues in the intra-phage RNA (B_eff^c and B_eff^u). The B_eff^u was close to that expected and increased from 70 to 130 as the temperature rose from 0 to 30°. The B_eff^c was much greater than that expected on the basis of the results with the monomer, suggesting that side reactions are involved in the inactivation of the phage at pH 5.0.A significant increase of the frequency of mutation occurs only under the conditions of predominant modification of cytidine (pH 5.0) at 0°. No such effect was observed at 30°. This was probably due to the increased contribution of inactivating side reactions. The effect of hydroxylamine on the phage under the conditions of predominant uridine modification (pH 8.0) did not lead to an increase in frequency of mutants.Incubation of the intact phage in acetate buffer resulted in considerable inactivation and mutations. Inactivation was inhibited by magnesium ions. Incubation at pH 5.5, of the phage inactivated by hydroxylamine treatment at pH 8.0, resulted in a considerable increase of the inefectivity with no effect on the frequency of mutants. The infectivity and the mutation frequency of the phage treated with hydroxylamine at pH 5.0 did not change as a result of incubation at pH 4.0 after the removal of the reagent.     

STUDIES ON THE PURIFICATION OF BACTERIOPHAGE

A simple method of concentrating and purifying bacteriophage has been described. The procedure consisted essentially in collecting the active agent on a reinforced collodion membrane of a porosity that would just retain all the active agent and permit extraneous material to pass through. Advantage was taken of the fact that B. coli will proliferate and regenerate bacteriophage in a completely diffusible synthetic medium with ammonia as the only source of nitrogen, which permitted the purification of the bacteriophage by copious washing. The material thus obtained was concentrated by suction and after thorough washing possessed all the activity of the original filtrate. It was labile, losing its activity in a few days on standing, and was quickly and completely inactivated upon drying. This material contained approximately 15 per cent of nitrogen and with 2 or 3 mg. samples of inactive dry residue it was possible to obtain positive protein color tests. The concentrated and purified bacteriophage has about 10^–14 mg. of nitrogen, or 6 x 10^–17 gm. of protein per unit of lytic activity. Assuming that each unit of activity represents a molecule, the calculated maximum average molecular weight would be approximately 36,000,000, and on the assumption of a spherical shape of particles and a density of 1.3, the calculated radius would be about 22 millimicra. By measurement of the diffusion rate, the average radius of particle of the fraction of the purified bacteriophage which diffuses most readily through a porous plate was found to be of the order of magnitude of 9 millimicra, or of a calculated molecular weight of 2,250,000. Furthermore, when this purified bacteriophage was fractionated by forcing it through a thin collodion membrane, which permits the passage of only the smaller particles, it was possible to demonstrate in the ultrafiltrate active particles of about 2 millimicra in radius, and of a calculated molecular weight of 25,000. It was of interest to apply this method of purification to a staphylococcus bacteriophage. Since this organism does not readily grow in synthetic medium, a diffusate of yeast extract medium was employed. The better of two preparations contained about 10^–12 mg. of nitrogen per unit of lytic activity. Although this is about one hundred times the amount of nitrogen found in an active unit of B. coli bacteriophage, nevertheless, the diffusion rate experiments gave results which paralleled those obtained with the coliphage. The diffusible particles of the crude staphylococcus bacteriophage had a radius of about 7 millimicra, and a calculated molecular weight of about 1,000,000, while the particles of the same phage which appeared in the ultrafiltrate through a thin collodion membrane had a radius of about 2.4 millimicra and a calculated molecular weight of about 45,000. It appears, therefore, that the active principle is distributed as particles of widely different sizes. However, since the smaller particles have all the properties of bacteriophage, the larger particles probably do not represent free molecules, but either are aggregates, or more likely, inactive colloids to which the active agent is adsorbed. The protein isolated, which bears the phage activity, is capable of stimulating the production of antilytic antibodies on parenteral injection into rabbits or guinea pigs. It retains its specific antigenicity when inactivated by formalin, but not when inactivated by drying.     

Orientation of the DNA in the filamentous bacteriophage f1

The filamentous bacteriophage f1 consists of a molecule of circular single-stranded DNA coated along its length by about 2700 molecules of the B protein. Five molecules of the A protein and five molecules of the D protein are located near or at one end of the virion, while ten molecules of the C protein are located near or at the opposite end. The two ends of the phage can be separated by reacting phage fragments, which have been generated by passage of intact phage through a French press, with antibody directed against the A protein (Grant et al., 1981a). By hybridizing the DNA isolated from either end of ³²P-labeled phage to specific restriction fragments of fl replicative form I DNA, we have determined that the single-stranded DNA of the filamentous bacteriophage f1 is oriented within the virion. For wild-type phage, the DNA that codes for the gene III protein is located at the A and D protein end and that which corresponds to the intergenic region is located close to the C protein end of the particle. The intergenic region codes for no protein but contains the origins for both viral and complementary strand DNA synthesis. Analysis of the DNA orientation in phage in which the plasmid pBR322 has been inserted into different positions within the intergenic region of fl shows that the C protein end of all sizes of filamentous phage particles appears to contain a common sequence of phage DNA. This sequence is located near the junction of gene IV and the intergenic region, and probably is important for normal packaging of phage DNA into infectious particles. There appears to be no specific requirement for the origins of viral and complementary strand DNA synthesis to be at the end of a phage particle.     

Change in the Template Specificity of RNA Phage SP-Replicase by the <Emphasis Type="Italic">E. coli</Emphasis> Cell Component

Qβ-REPLICASE was isolated from E. coli infected with the RNA bacteriophage Qβ as RNA-dependent RNA polymerase which had template specificity¹. RNA phage SP², which is distinct from RNA phages isolated previously^3,4, has been isolated in our laboratory and SP-replicase⁵ was purified from E. coli infected with SP-phage. SP-replicase has a template specificity different from that of Qβ-replicase. By using this new RNA-replicase, comparison between two distinct replicases has become possible.     

Use of mini-antibodies for detection of bacteriophages by the electroaucoustic analysis method

The possibility of detecting bacteriophages using phage mini-antibodies by the electroacoustic analysis method using bacteriophages FA1-59b was shown. It was found that the frequency dependence of the real and imaginary parts of the electrical impedance of a resonator with a suspension of phages and the appropriate antibodies significantly differs from that of the resonator with a control virus suspension without addition of mini-antibodies. The amount of FAl-Sp59b bacteriophage in the analyzed suspension varied from ~10¹⁰ to 10⁶ phage/mL; the analysis did not take longer than 5 min. The change in the real or imaginary parts of the electrical impedance at the fixed frequency near the resonance after addition of specific mini-antibodies in the suspension appeared to be an optimal information parameter to obtain reliable information. These results may allow the development of a biological sensor to identify and quantify viruses in the liquid phase.     

Bacteriophage MB78 DNA synthesis is specifically inhibited by the chelating agent ethylene diamine tetraacetic acid

The growth of bacteriophage MB78, a virulent phage of Salmonella typhimurium is extremely sensitive to the chelating agent EDTA. Other chelating agents like EGTA, a specific chelator for Ca²⁺ and orthophenanthroline which chelates Zn²⁺ and Fe²⁺ have no effect. EDTA stops phage MB78 DNA synthesis while synthesis of host DNA and other Salmonella phage DNA are not affected in presence of such low concentrations of EDTA. The present report indicates that some early phage function(s) and most probably the phage DNA synthesis are sensitive to EDTA which is probably due to chelation of Mg²⁺.     

Reduction in Exopolysaccharide Viscosity as an Aid to Bacteriophage Penetration through Pseudomonas aeruginosa Biofilms

To cause an infection, bacteriophages must penetrate the alginate exopolysaccharide of Pseudomonas aeruginosa to reach the bacterial surface. Despite a lack of intrinsic motility, phage were shown to diffuse through alginate gels at alginate concentrations up to 8% (wt/vol) and to bring about a 2-log reduction in the cell numbers in 20-day-old biofilms of P. aeruginosa. The inability of alginate to act as a more effective diffusional barrier suggests that phage may cause a reduction in the viscosity of the exopolysaccharide. Samples (n = 5) of commercial alginate and purified cystic fibrosis (CF) alginate were incubated with 2 × 10⁸ purified phage per ml for 24 h at 37°C. After incubation the samples and controls were subjected to rheological analysis with a Carrimed controlled stress rheometer. The viscosities of phage-treated samples were reduced by up to 40% compared to those of controls incubated in the absence of phage. The experiment was repeated by using phage concentrations of 10¹⁰ and 10¹² phage per ml and samples taken for analysis at intervals up to 4 h. The results indicated that there was a time- and concentration-dependent reduction in viscosity of up to 40% compared to the viscosities of the controls. Commercial and purified CF alginate samples, both phage treated and untreated, were subjected to gel filtration chromatography by using Sephacryl High Resolution S-400 medium in order to obtain evidence of degradation. The results demonstrated that alginate treated with phage had a lower molecular weight than untreated alginate. The data suggest that bacteriophage migration through P. aeruginosa biofilms may be facilitated by a reduction in alginate viscosity brought about by enzymic degradation and that the source of the enzyme may be the bacterial host itself.     

Isolation and Partial Characterization of a Virulent Bacteriophage IHQ1 Specific for Aeromonas punctata from Stream Water

Aeromonas punctata is the causative agent of septicemia, diarrhea, wound infections, meningitis, peritonitis, and infections of the joints, bones and eyes. Bacteriophages are often considered alternative agents for controlling bacterial infection and contamination. In this study, we described the isolation and preliminary characterization of bacteriophage IHQ1 (family Myoviridae) active against the Gram-negative bacterial strain A. punctata. This virulent bacteriophage was isolated from stream water sample. Genome analysis indicated that phage IHQ1 was a double-stranded DNA virus with an approximate genome size of 25–28 kb. The initial characterization of this newly isolated phage showed that it has a narrow host range and infects only A. punctata as it failed to infect seven other clinically isolated pathogenic strains, i.e., methicillin-resistant Staphylococcus aureus 6403, MRSA 17644, Acinetobacter 33408, Acinetobacter 1172, Pseudomonas aeruginosa 22250, P. aeruginosa 11219, and Escherichia coli. Proteomic pattern of phage IHQ1, generated by SDS-PAGE using purified phage particles, showed three major and three minor protein bands with molecular weights ranging from 25 to 70 kDa. The adsorption rate of phage IHQ1 to the host bacterium was also determined, which was significantly enhanced by the addition of 10 mM CaCl₂. From the single-step growth experiment, it was inferred that the latent time period of phage IHQ1 was 24 min and a burst size of 626 phages per cell. Moreover, the pH and thermal stability of phage IHQ1 were also investigated. The maximum stability of the phage was observed at optimal pH 7.0, and it was totally unstable at extreme acidic pH 3; however, it was comparatively stable at alkaline pH 11.0. At 37°C the phage showed maximum number of plaques, and the viability was almost 100%. The existence of Aeromonas bacteriophage is very promising for the eradication of this opportunistic pathogen and also for future applications such as the design of new detection and phage typing (diagnosis) methods. The specificity of the bacteriophage for A. punctata makes it an attractive candidate for phage therapy of A. punctata infections.     

Dual Functions of Bacteriophage T4D Gene 28 Product: Structural Component of the Viral Tail Baseplate Central Plug and Cleavage Enzyme for Folyl Polyglutamates II. Folate Metabolism and Polyglutamate Cleavage Activity of Uninfected and Infected Escherichia coli Cells and Bacteriophage Particles

We investigated the role of the T4D bacteriophage gene 28 product in folate metabolism in infected Escherichia coli cells by using antifolate drugs and a newly devised assay for folyl polyglutamate cleavage activity. Preincubation of host E. coli cells with various sulfa drugs inhibited phage production by decreasing the burst size when the phage particles produced an altered gene 28 product (i.e., after infection under permissive conditions with T4D 28^ts or T4D am28). In addition, we found that another folate analog, pyrimethamine, also inhibited T4D 28^ts production and T4D 28am production, but this analog did not inhibit wild-type T4D production. A temperature-resistant revertant of T4D 28^ts was not sensitive to either sulfa drugs or pyrimethamine. We developed an assay to measure the enzymatic cleavage of folyl polyglutamates. The high-molecular-weight folyl polyglutamate substrate was isolated from E. coli B cells infected with T4D am28 in the presence of labeled glutamic acid and was characterized as a folate compound containing 12 to 14 labeled glutamate residues. Extracts of uninfected bacteria liberated glutamate residues from this substrate with a pH optimum of 8.4 to 8.5. Extracts of bacteriophage T4D-infected E. coli B cells exhibited an additional new folyl polyglutamate cleavage activity with a pH optimum of about 6.4 to 6.5, which was clearly distinguished from the preexisting activity in the uninfected host cells. This new activity was induced in E. coli B cells by infection with wild-type T4D and T4D amber mutants 29⁻, 26⁻, 27⁻, 51⁻, and 10⁻, but it was not induced under nonpermissive conditions by T4D am28 or by T4D 28^ts. Mutations in gene 28 affected the properties of the induced cleavage enzyme. Wild-type T4D-induced cleavage activity was not inhibited by pyrimethamine, whereas the T4D 28^ts activity induced at a permissive temperature was inhibited by this folate analog. Folyl polyglutamate cleavage activity characteristic of the activity induced in host cells by wild-type T4D or by T4D gene 28 mutants was also found in highly purified preparations of these phage ghost particles. The T4D-induced cleavage activity could be inhibited by antiserum prepared against highly purified phage baseplates. We concluded that T4D infection induced the formation of a new folyl polyglutamate cleavage enzyme and that this enzyme was coded for by T4D gene 28. Furthermore, since this gene product was a baseplate tail plug component which had both its antigenic sites and its catalytic sites exposed on the phage particle, it was apparent that this enzyme formed part of the distal surface of the phage baseplate central tail plug.     

Structure and Functions of the Bacteriophage P22 Tail Protein

The product of gene 9 (gp9) of Salmonella typhimurium bacteriophage P22 is a multifunctional structural protein. This protein is both a specific glycosidase which imparts the adsorption characteristics of the phage for its host and a protein which participates in a specific assembly reaction during phage morphogenesis. We have begun a detailed biochemical and genetic analysis of this gene product. A relatively straightforward purification of this protein has been devised, and various physical parameters of the protein have been determined. The protein has an s_20,w of 9.3S, a D_20,w of 4.3 × 10⁻⁷ cm²/s, and a molecular weight, as determined by sedimentation equilibrium, of 173,000. The purified protein appears as a prolate ellipsoid upon electron microscopic examination, with an axial ratio of 4:1, which is similar to the observed shape when it is attached to the phage particle. The molecular weight is consistent with the tail protein being a dimer of gp9 and each phage containing six of these dimers. An altered form of the tail protein has been purified from supF cells infected with a phage strain carrying an amber mutation in gene 9. Phage “tailed” with this altered form of gp9 adsorb to susceptible cells but form infectious centers with a severely reduced efficiency (ca. 1%). Biochemical analysis of the purified wild-type and genetically altered tail proteins suggests that loss of infectivity correlates with a loss in the glycosidase activity of the protein (2.5% residual activity). From these results we propose that the glycosidic activity of the P22 tail protein is not essential for phage assembly or adsorption of the phage to its host but is required for subsequent steps in the process of infection.     

Bacteriophage Therapy To Reduce Campylobacter jejuni Colonization of Broiler Chickens

Colonization of broiler chickens by the enteric pathogen Campylobacter jejuni is widespread and difficult to prevent. Bacteriophage therapy is one possible means by which this colonization could be controlled, thus limiting the entry of campylobacters into the human food chain. Prior to evaluating the efficacy of phage therapy, experimental models of Campylobacter colonization of broiler chickens were established by using low-passage C. jejuni isolates HPC5 and GIIC8 from United Kingdom broiler flocks. The screening of 53 lytic bacteriophage isolates against a panel of 50 Campylobacter isolates from broiler chickens and 80 strains isolated after human infection identified two phage candidates with broad host lysis. These phages, CP8 and CP34, were orally administered in antacid suspension, at different dosages, to 25-day-old broiler chickens experimentally colonized with the C. jejuni broiler isolates. Phage treatment of C. jejuni-colonized birds resulted in Campylobacter counts falling between 0.5 and 5 log₁₀ CFU/g of cecal contents compared to untreated controls over a 5-day period postadministration. These reductions were dependent on the phage-Campylobacter combination, the dose of phage applied, and the time elapsed after administration. Campylobacters resistant to bacteriophage infection were recovered from phage-treated chickens at a frequency of <4%. These resistant types were compromised in their ability to colonize experimental chickens and rapidly reverted to a phage-sensitive phenotype in vivo. The selection of appropriate phage and their dose optimization are key elements for the success of phage therapy to reduce campylobacters in broiler chickens.     

噬菌体gp17基因2种重组产物的抗菌效应比较

通过重组技术获得大肠埃希菌噬菌体内溶素纯化蛋白和表面展示噬菌体,并观察产物的生物效应。将肠侵袭性大肠埃希菌EIEC 8401噬菌体LSB-1内溶素基因gp17构建到质粒pET300中,并在大肠埃希菌BL21中诱导表达,通过Ni柱纯化系统纯化产物;利用噬菌体展示技术构建T7-LSB-gp17重组噬菌体,通过双层琼脂法纯化噬菌体,并观察2种产物的抗菌效应。2 139 bp的gp17基因通过重组技术表达出78.3 ku的可溶性蛋白,纯化后浓度为2.38 mg/mL,其对EIEC8401有良好的抑菌活性,但对其他试验菌无抗性;通过噬菌体展示技术构建的重组噬菌体T7-LSB-gp17通过SDS-PAGE电泳显示在78 ku处有表达增强,对EIEC8401无感染、裂解作用,但对EIEC8401及其他试验菌有明显溶菌作用,宿主谱增加。通过重组技术获得的噬菌体LSB-1内溶素基因gp17的产物对LSB-1噬菌体原宿主具有明显的抑制效应。其中gp17表达的纯化蛋白具有明显的宿主专一性,重组噬菌体悬液有较宽种类的抗菌作用。这可能是因为gp17蛋白与噬菌体表面复杂空间结构的相互作用产生的生物效应。     

Protein Components of Bacteriophages λ and λ Virulent

Parallel studies have been made of the protein coats of the temperate bacteriophage λ and of a deletion mutant, λ virulent. A new method for preparing ghosts of both phages by the action of Cu⁺⁺ is described. Protein ghosts of both phages can be dissolved in citrate at pH values below 3, more rapidly in the presence of 8 m urea. Both phages yielded three apparently identical protein components which can be separated by thin-layer gel filtration and thin-layer gel electrophoresis. The protein of molecular weight 47,000 ± 1,500 represents about 55% of the protein of the ghosts and is therefore likely to be the subunit of the head. The other proteins of molecular weight 30,000 ± 1,500 and 16,000 ± 1,500 represent approximately 25% and 20% of the protein, respectively. Amino acid analyses of the ghosts from the two phages have been carried out and show no significant differences. The buoyant density of phage λ virulent is 0.016 g/ml less than that of λ. Since no differences have been found in the protein components of the two phages, this indicates that the virulent mutant contains approximately 16% less deoxyribonucleic acid than the temperate phage.     

Differences in susceptibility to restriction by E. coli B between various heteroduplex molecules of bacteriophage FD DNA

Fragments of B-modified bacteriophage fd sB1^o sB2 RF DNA were prepared with the help of purified endonuclease R from Haemophilus parainfluenzae (Hpa II). These were hybridized with unmodified circular single stranded fd DNA. The resulting partial heteroduplex molecules were assayed for infectivity on competent cells of B-restricting and non restricting strains of E. coli. There of such heteroduplexes originating from neighbouring fragments on the physical map of fd RF DNA were shown to be more resistant to Eco B restriction than six others and the unmodified control. It is suggested that the three corresponding vicinal fragments contain essential parts of the Eco B recognition site on this phage DNA.     

Construction and properties of a recombinant plasmid containing gene 32 of bacteriophage T4D

This paper describes the construction and characterization of a chimeric plasmid that encodes the single-stranded DNA-binding protein of bacteriophage T4D (the product of gene 32). The plasmid contains a 2·6 × 10³ base HindIII segment of T4 DNA that includes genes 59 and 32 as well as a portion of gene 33. Isolation of bacteria carrying the recombinant plasmid became possible when the segment of phage DNA contained an amber mutation in gene 32. This suggests that a functional gene 32 is deleterious to the cell. Using antibody to gene 32 protein, we have been able to demonstrate expression of the plasmid-borne gene 32 in uninfected bacteria. Deletion variants of the gene 32 plasmid have been constructed in vitro. These have been used to align the genetic map of the region with the restriction map and to study phage gene expression from the plasmid in both infected and uninfected cells. In phage-infected cells the level of functional gene 32 product regulates the efficiency of translation of its own messenger RNA. We also observe such self-regulation for gene 32 present on the plasmid.     

Translation of bacteriophage R17 and Qbeta RNA in a mammalian cell-free system

The polycistronic RNAs from both bacteriophage R17 and Qβ are translated in a mammalian cell-free system of purified and partially purified components. The requirement of one of the partially purified initiation factors (IF-E₃ from rabbit reticulocytes) for the phage RNA translation is strikingly different from that for rabbit globin messenger RNA translation. The phage RNA-directed products are characterized by acrylamide gel electrophoresis and compared with those synthesized in an Escherichia coli cell-free system. There is good agreement between the respective coat proteins and the presumptive synthetase proteins. R17 RNA directs the synthesis of two additional defined polypeptides. However, their possible relationship with the A-protein cistron has not yet been investigated. The RNA from the amB2 mutant of R17, which carries an amber triplet at position 6 in the coat protein cistron, directs the synthesis of the same polypeptides as the wild-type RNA with the exception of the coat protein which is completely abolished. This identifies the product made with wild-type RNA as coat protein and provides a direct in vitro assay for the suppression of nonsense mutations in eukaryotic cells.     

Role of genes 46 and 47 in bacteriophage T4 reproduction: III. Formation of joint molecules in biparental recombination

The role of bacteriophage T4 gene 46 in recombination between non-replicating chromosomes was examined. DNA was extracted from Escherichia coli B infected with a mixture of [³H]thymidine-labeled and (¹³C, ¹⁵N)-labeled T4 multiple mutants under non-permissive conditions. The densities of extracted, purified DNAs were determined by neutral cesium sulfate density-gradient centrifugation. When the phage was a double mutant defective in both DNA ligase and DNA polymerase genes, a considerable portion of the ³H label was found at a hybrid density. By contrast, when phage had a third mutation in gene 46, the amount of ³H label found at the hybrid position was greatly reduced. These findings indicate that hybrid molecule formation requires the function of gene 46.     

Tapping diversity lost in transformations—in vitro amplification of ligation reactions

Molecular evolution is a powerful means of engineering proteins. It usually requires the generation of a large recombinant DNA library of variants for cloning into a phage or plasmid vector, and the transformation of a host organism for expression and screening of the variant proteins. However, library size is often limited by the low yields of circular DNA and the poor transformation efficiencies of linear DNA. Here we have overcome this limitation by amplification of recombinant circular DNA molecules directly from ligation reactions. The amplification by bacteriophage Phi29 polymerase increased the number of transformants; thus from a nanogram-scale ligation of DNA fragments comprising two sub-libraries of variant antibody domains, we succeeded in amplifying a highly diverse and large combinatorial phage antibody library (>10⁹ transformants in Escherichia coli and 10⁵-fold more transformants than without amplification). From the amplified library, but not from the smaller un-amplified library, we could isolate several antibody fragments against a target antigen. It appears that amplification of ligations with Phi29 polymerase can help recover clones and molecular diversity otherwise lost in the transformation step. A further feature of the method is the option of using PCR-amplified vectors for ligations.