Chest health surveillance utility in the early detection of bronchiolitis obliterans syndrome in children after allo-SCT.

To prospectively assess whether periodic chest health surveillance is beneficial for the early detection of bronchiolitis obliterans syndrome (BOS) in children after allo-SCT. Children up to 18 years of age receiving allo-SCT from September 2009 to September 2011 were included. Surveillance consisted of the following: a 7-item respiratory system questionnaire of cough, wheeze and shortness of breath; focused physical examination; and pulmonary function test (PFT) conducted before SCT and at 1, 3, 6, 9, 12, 18 and 24 months after SCT. Thirty-nine patients were enrolled. Five children developed BOS at a median time of 192 days (range 94-282). Positive response comparisons between the BOS group vs the non-BOS group were NS for history questionnaire (P=0.2), heart rate (P=0.3), respiratory rate (P=0.3) and oxygen saturation monitoring (P=0.8). Differences between the two groups for chest auscultation and PFT were statistically significant (P=0.03 and P=0.01, respectively). However, chest auscultation in the BOS group was only positive after BOS diagnosis. PFT reduction was evident in the asymptomatic phase (BOS group 33%; non-BOS group 4.5%, P=0.01). Changes in PFT, but not history/physical examination, allow the early detection of BOS in children after SCT. Our study is limited by the small sample size.

Microfibril-associated protein 4 binds to surfactant protein A (SP-A) and colocalizes with SP-A in the extracellular matrix of the lung.

Pulmonary surfactant protein A (SP-A) is an oligomeric collectin that recognizes lipid and carbohydrate moieties present on broad range of micro-organisms, and mediates microbial lysis and clearance. SP-A also modulates multiple immune-related functions including cytokine production and chemotaxis for phagocytes. Here we describe the molecular interaction between the extracellular matrix protein microfibril-associated protein 4 (MFAP4) and SP-A. MFAP4 is a collagen-binding molecule containing a C-terminal fibrinogen-like domain and a N-terminal located integrin-binding motif. We produced recombinant MFAP4 with a molecular mass of 36 and 66 kDa in the reduced and unreduced states respectively. Gel filtration chromatography and chemical crosslinking showed that MFAP4 forms oligomers of four dimers. We demonstrated calcium-dependent binding between MFAP4 and human SP-A1 and SP-A2. No binding was seen to recombinant SP-A composed of the neck region and carbohydrate recognition domain of SP-A indicating that the interaction between MFAP4 and SP-A is mediated via the collagen domain of SP-A. Monoclonal antibodies directed against MFAP4 and SP-A were used for immunohistochemical analysis, which demonstrates that the two molecules colocalize both on the elastic fibres in the interalveolar septum and in elastic lamina of pulmonary arteries of chronically inflamed lung tissue. We conclude, that MFAP4 interacts with SP-A via the collagen region in vitro, and that MFAP4 and SP-A colocates in different lung compartments indicating that the interaction may be operative in vivo.

Alveolar macrophage deficiency in osteopetrotic mice deficient in macrophage colony-stimulating factor is spontaneously corrected with age and associated with matrix metalloproteinase expression and emphysema.

Macrophage colony-stimulating factor (M-CSF) is one of several hematologic growth factors capable of regulating the survival, proliferation, and differentiation of macrophages, but its role in modulation of the accumulation and function of alveolar macrophages (AMs) in vivo is not well defined. Osteopetrotic (Op/Op) mice have no detectable M-CSF and show variable tissue-specific reductions in macrophage numbers. It was hypothesized that AMs would be decreased in number and have altered function in Op/Op mice because of the absence of M-CSF. Lung macrophages identified by Mac-3 staining in lung sections were decreased in number in 20-day-old Op/Op mice (P <.001) but not Op/Op mice older than 4 months (P =.68) compared with findings in age-matched littermate controls. The numbers of AMs recovered by bronchoalveolar lavage (BAL) were also reduced in young but not adult Op/Op mice compared with controls. Expression of interleukin-3 (IL-3) was increased in the lungs of Op/Op mice compared with controls as determined by quantification of IL-3 cytokine levels (P =.04), bioactivity (P =.02), and messenger RNA transcript levels. AMs of Op/Op mice spontaneously released higher levels of matrix metalloproteinases (MMPs) than AMs of controls as determined by immunohistochemical staining of AMs and zymographic assessment of BAL fluid and AM lysates. Consistent with an increased release of MMP, Op/Op mice had abnormal elastin deposition and spontaneously developed emphysema in the absence of molecular or cellular evidence of lung inflammation. These data show that the AM deficiency observed in young Op/Op mice is spontaneously corrected with age and is associated with increased lung levels of IL-3, spontaneous MMP expression by AMs, and destruction of lung tissue.

The collagen-like region of surfactant protein A (SP-A) is required for correction of surfactant structural and functional defects in the SP-A null mouse.

Pulmonary surfactant isolated from gene-targeted surfactant protein A null mice (SP-A(-/-)) is deficient in the surfactant aggregate tubular myelin and has surface tension-lowering activity that is easily inhibited by serum proteins in vitro. To further elucidate the role of SP-A and its collagen-like region in surfactant function, we used the human SP-C promoter to drive expression of rat SP-A (rSPA) or SP-A containing a deletion of the collagen-like domain (DeltaG8-P80) in the Clara cells and alveolar type II cells of SP-A(-/-) mice. The level of the SP-A in the alveolar wash of the SP-A(-/-,rSP-A) and SP-A(-/-,DeltaG8-P80) mice was 6.1-and 1.3-fold higher, respectively, than in the wild type controls. Tissue levels of saturated phosphatidylcholine were slightly reduced in the SP-A(-/-,rSP-A) mice compared with SP-A(-/-) littermates. Tubular myelin was present in the large surfactant aggregates isolated from the SP-A(-/-,rSP-A) lines but not in the SP-A(-/-,DeltaG8-P80) mice or SP-A(-/-) controls. The equilibrium and minimum surface tensions of surfactant from the SP-A(-/-,rSP-A) mice were similar to SP-A(-/-) controls, but both were markedly elevated in the SP-A(-/-,DeltaG8-P80) mice. There was no defect in the surface tension-lowering activity of surfactant from SP-A(+/+,DeltaG8-P80) mice, indicating that the inhibitory effect of DeltaG8-P80 on surface activity can be overcome by wild type levels of mouse SP-A. The surface activity of surfactant isolated from the SP-A(-/-,rSP-A) but not the SP-A(-/-,DeltaG8-P80) mice was more resistant than SP-A(-/-) littermate control animals to inhibition by serum proteins in vitro. Pressure volume relationships of lungs from the SP-A(-/-), SP-A(-/-,rSP-A), and SP-A(-/-,DeltaG8-P80) lines were very similar. These data indicate that expression of SP-A in the pulmonary epithelium of SP-A(-/-) animals restores tubular myelin formation and resistance of isolated surfactant to protein inhibition by a mechanism that is dependent on the collagen-like region.

Domains of surfactant protein A that affect protein oligomerization, lipid structure and surface tension.

Surfactant protein A (SP-A) is an abundant protein found in pulmonary surfactant which has been reported to have multiple functions. In this review, we focus on the structural importance of each domain of SP-A in the functions of protein oligomerization, the structural organization of lipids and the surface-active properties of surfactant, with an emphasis on ultrastructural analyses. The N-terminal domain of SP-A is required for disulfide-dependent protein oligomerization, and for binding and aggregation of phospholipids, but there is no evidence that this domain directly interacts with lipid membranes. The collagen-like domain is important for the stability and oligomerization of SP-A. It also contributes shape and dimension to the molecule, and appears to determine membrane spacing in lipid aggregates such as common myelin and tubular myelin. The neck domain of SP-A is primarily involved in protein trimerization, which is critical for many protein functions, but it does not appear to be directly involved in lipid interactions. The globular C-terminal domain of SP-A clearly plays a central role in lipid binding, and in more complex functions such as the formation and/or stabilization of curved membranes. In recent work, we have determined that the maintenance of low surface tension of surfactant in the presence of serum protein inhibitors requires cooperative interactions between the C-terminal and N-terminal domains of the molecule. This effect of SP-A requires a high degree of oligomeric assembly of the protein, and may be mediated by the activity of the protein to alter the form or physical state of surfactant lipid aggregates.

Formation of folds and vesicles by dipalmitoylphosphatidylcholine monolayers spread in excess.

Lipid monolayers exist in several biological systems, including the stratum corneum of the skin, the fluid tear film of the eye, the Eustachian tube of the ear, and airway and alveolar pulmonary surfactants. In this paper, the monolayer-to-bilayer transition was studied using dipalmitoylphosphatidylcholine (DPPC) as the model. Depositing DPPC organic solvent solutions in excess at an air:buffer interface led to the formation of elongated structures which could be imaged on carbon grids by transmission electron microscopy. The structures appeared to be DPPC folds protruding into the sol. The structures were frequently ordered with respect to one another, suggesting that they arose during lateral compression due to excess DPPC and are characteristic of a type of monolayer collapse phase. In some cases, series of short folds in an extended line and series of vesicles in line or parallel to the folds were observed. This suggests the elongated folds are unstable and can resolve by forming vesicles. Fold formation occurred at defined lipid concentrations above which more vesicles were observed. Surfactant protein-A did not influence fold or vesicle formation but bound to the edges of these structures preferentially. It is concluded that DPPC monolayers can form bilayers spontaneously in the absence of surfactant apoproteins, other proteins or agents.

Myelin basic protein component C1 in increasing concentrations can elicit fusion, aggregation, and fragmentation of myelin-like membranes.

Myelin basic protein (MBP) is considered to have a primary role in the formation and maintenance of the myelin sheath. Many studies using artificial vesicle systems of simple lipid composition, and generally small size, have shown that MBP can elicit vesicle fusion, aggregation, or even fragmentation under different conditions. Here, we have studied the effects of increasing concentrations of bovine MBP charge isomer C1 (MBP/C1) on large unilamellar vesicles (LUVs) composed of phosphatidylcholine and phosphatidylserine (92:8 molar ratio), or with a lipid composition similar to that of the myelin membrane in vivo (Cyt-LUVs). Using absorbance spectrophotometry, fluorescence resonance energy transfer, dynamic light scattering and transmission electron microscopy, we have shown that vesicle aggregation and some vesicle fusion occurred upon addition of MBP/C1, and as the molar protein-lipid ratio increased. Fragmentation of Cyt-LUVs was observed at very high protein concentrations. These results showed that the phenomena of vesicle fusion, aggregation, and fragmentation can all be observed in one in vitro system, but were dependent on lipid composition and on the relative proportions of protein and lipid.

Three-dimensional structure of rat surfactant protein A trimers in association with phospholipid monolayers.

Surfactant protein A (SP-A) is a C-type lectin found primarily in the lung and plays a role in innate immunity and the maintenance of surfactant integrity. To determine the three-dimensional (3D) structure of SP-A in association with a lipid ligand, we have used single particle electron crystallography and computational 3D reconstruction in combination with molecular modeling. Recombinant rat SP-A, containing a deletion of the collagen-like domain, was incubated with dipalmitoylphosphatidylcholine:egg phosphatidylcholine (1:1, wt/wt) lipid monolayers in the presence of calcium, negatively stained, and examined by transmission electron microscopy. Images of SP-A-lipid complexes with different angular orientations were used to reconstruct the 3D structure of the protein. These results showed that SP-A subunits readily formed trimers and interacted with lipid monolayers exclusively via the globular domains. A homology-based molecular model of SP-A was generated and fitted into the electron density map of the protein. The plane of the putative lipid-protein interface was relatively flat and perpendicular to the hydrophobic neck region, and the cleft region in the middle of the trimer had no apparent charge clusters. Amino acid residues that are known to affect lipid interactions, Glu(195) and Arg(197), were located at the protein-lipid interface. The molecular model indicated that the hydrophobic neck region of the SP-A did not interact with lipid monolayers but was instead involved in intratrimeric subunit interactions. The glycosylation site of SP-A was located at the side of each subunit, suggesting that the covalently linked carbohydrate moiety probably occupies the spaces between the adjacent globular domains, a location that would not sterically interfere with ligand binding.

Cryoelectron microscopy of protein-lipid complexes of human myelin basic protein charge isomers differing in degree of citrullination.

Myelin basic protein (MBP) is considered to be essential for the maintenance of stability of the myelin sheath. Reduction in cationicity of MBP, especially due to conversion of positively charged arginine residues to uncharged citrulline (Cit), has been found to be associated with multiple sclerosis (MS). Here, the interactions of an anionic phosphatidylserine/monosialoganglioside-G(M1) (4:1, w:w) lipid monolayer with 18.5-kDa MBP preparations from age-matched adult humans without MS (no Cit residues), with chronic MS (6 Cit), and with acute Marburg-type MS (18 Cit) were studied by transmission and ultralow dose scanning transmission electron microscopy under cryogenic conditions. Immunogold labeling and single particle electron crystallography were used to define the nature of the complexes visualized. These electron microscopical analyses showed that the three different MBP charge isomers all formed uniformly sized and regularly shaped protein-lipid complexes with G(M1), probably as hexamers, but exhibited differential association with and organization of the lipid. The least cationic Marburg MBP-Cit(18) formed the most open protein-lipid complex. The data show a disturbance in lipid-MBP interactions at the ultrastructural level that is related to degree of citrullination, and which may be involved in myelin degeneration in multiple sclerosis.

DNA binding and aggregation properties of the vaccinia virus I3L gene product.

The vaccinia virus I3L gene encodes a single-stranded DNA-binding protein which may play a role in viral replication and genetic recombination. We have purified native and recombinant forms of gpI3L and characterized both the DNA-binding reaction and the structural properties of DNA-protein complexes. The purified proteins displayed anomalous electrophoretic properties in the presence of sodium dodecyl sulfate, behaving as if they were 4-kDa larger than the true mass. Agarose gel shift analysis was used to monitor the formation of complexes composed of single-stranded DNA plus gpI3L protein. These experiments detected two different DNA binding modes whose formation was dependent upon the protein density. The transition between the two binding modes occurred at a nucleotide to protein ratio of about 31 nucleotides per gpI3L monomer. S1 nuclease protection assay revealed that at saturating protein densities, each gpI3L monomer occludes 9.5 +/- 2.5 nucleotides. In the presence of magnesium, gpI3L promoted the formation of large DNA aggregates from which double-stranded DNA was excluded. Electron microscopy showed that, in the absence of magnesium and at low protein densities, gpI3L forms beaded structures on DNA. At high protein density the complexes display a smoother and less compacted morphology. In the presence of magnesium the complexes contained long fibrous and tangled arrays. These results suggest that gpI3L can form octameric complexes on DNA much like those formed by Escherichia coli single-stranded DNA protein. Moreover, the capacity to aggregate DNA may provide an environment in which hybrid DNA formation could occur during DNA replication.

Filaments of surfactant protein A specifically interact with corrugated surfaces of phospholipid membranes.

Pulmonary surfactant, a mixture of lipids and surfactant proteins (SPs), plays an important role in respiration and gas exchange. SP-A, the major SP, exists as an octadecamer that can self-associate to form elongated protein filaments in vitro. We have studied here the association of purified bovine SP-A with lipid vesicle bilayers in vitro with negative staining with uranyl acetate and transmission electron microscopy. Native bovine surfactant was also examined by transmission electron microscopy of thinly sectioned embedded material. Lipid vesicles made from dipalmitoylphosphatidylcholine and egg phosphatidylcholine (1:1 wt/wt) generally showed a smooth surface morphology, but some large vesicles showed a corrugated one. On the smooth-surfaced vesicles, SP-As primarily interacted in the form of separate octadecamers or as multidirectional protein networks. On the surfaces of the striated vesicles, SP-As primarily formed regularly spaced unidirectional filaments. The mean spacing between adjacent striations and between adjacent filaments was 49 nm. The striated surfaces were not essential for the formation of filaments but appeared to stabilize them. In native surfactant preparations, SP-A was detected in the dense layers. This latter arrangement of the lipid bilayer-associated SP-As supported the potential relevance of the in vitro structures to the in vivo situation.

Formation of membrane lattice structures and their specific interactions with surfactant protein A.

Biological membranes exist in many forms, one of which is known as tubular myelin (TM). This pulmonary surfactant membranous structure contains elongated tubes that form square lattices. To understand the interaction of surfactant protein (SP) A and various lipids commonly found in TM, we undertook a series of transmission-electron-microscopic studies using purified SP-A and lipid vesicles made in vitro and also native surfactant from bovine lung. Specimens from in vitro experiments were negatively stained with 2% uranyl acetate, whereas fixed native surfactant was delipidated, embedded, and sectioned. We found that dipalmitoylphosphatidylcholine-egg phosphatidylcholine (1:1 wt/wt) bilayers formed corrugations, folds, and predominantly 47-nm-square latticelike structures. SP-A specifically interacted with these lipid bilayers and folds. We visualized other proteolipid structures that could act as intermediates for reorganizing lipids and SP-As. Such a reorganization could lead to the localization of SP-A in the lattice corners and could explain, in part, the formation of TM-like structures in vivo.

Marburg's variant of multiple sclerosis correlates with a less compact structure of myelin basic protein.

Multiple sclerosis (MS) is an autoimmune disease in which the myelin sheath of the central nervous system is degraded, and the 18.5 kDa isoform of myelin basic protein (MBP) is reduced in cationicity. In a unique case of acute, fulminating MS (Marburg's variant), MBP is considerably less cationic than MBP from both normal, and chronic MS-afflicted individuals. This electron microscopical study has identified that, in vitro, the less cationic Marburg MBP isomer forms a more extended protein-lipid complex than MBP from healthy or chronic MS-afflicted individuals. This correlation implies that chemical modifications to MBP in vivo contribute directly to the structural instability of myelin, and subsequent autoantigenic presentation of this protein, observed in vivo in MS.

Shope fibroma virus DNA topoisomerase catalyses holliday junction resolution and hairpin formation in vitro.

The telomeres of poxviral chromosomes comprise covalently closed hairpin structures bearing mismatched bases. These hairpins are formed as concatemeric replication intermediates and are processed into mature, unit-length genomes. The structural transitions and enzymes involved in telomere resolution are poorly understood. Here we show that the type I topoisomerase of Shope fibroma virus (SFV) can promote a recombination reaction which converts cloned SFV replication intermediates into hairpin-ended molecules resembling mature poxviral telomeres. Recombinant SFV topoisomerase linearised a palindromic plasmid bearing 1.5 kb of DNA encoding the SFV concatemer junction, at a site near the centre of inverted-repeat symmetry. Most of these linear reaction products bore hairpin tips as judged by denaturing gel electrophoresis. The resolution reaction required palindromic SFV DNA sequences and was inhibited by compounds which block branch migration (MgCl2) or poxviral topoisomerases. The resolution reaction was also slow, needed substantial quantities of topoisomerase, and required that the palindrome be extruded in a cruciform configuration. DNA cleavage experiments identified a pair of suitably oriented topoisomerase recognition sites, 90 bases from the centre of the cloned SFV terminal inverted repeat, which may mark the resolution site. These data suggest a resolution scheme in which branch migration of a Holliday junction through a site occupied by covalently bound topoisomerase molecules, could lead to telomere resolution.

Cation-mediated conformational variants of surfactant protein A.

Surfactant protein A (SP-A) is the major protein of pulmonary surfactant. This protein is implicated in regulating surfactant secretion, alveolar processing, recycling, and in non-serum-induced immune response. An increasing body of work indicates the importance of cations, particularly calcium, on SP-A function. However, little information exists on the effects of cations on SP-A quaternary structure. Here, the quaternary organisation of bovine surfactant protein A in the presence of cations has been quantitatively and systematically studied by transmission electron microscopy. The conformation of SP-A is altered by the presence of cations, especially calcium, then sodium, and to a small extent, magnesium. There is a transition concentration, unique for each cation, at which a conformational switch occurs. These transition concentrations are: 5 mM for CaCl2, 100 mM for NaCl and 1 mM for MgCl2. Below these concentrations, SP-A exists primarily in an opened form with a large head diameter of 20 nm; above it, SP-A is mostly in a closed form due to a compaction of the headgroups resulting in a head diameter of 11 nm. There is a corresponding increase in particle length from 17 nm for opened SP-A to 20 nm for closed SP-A. The fact that the transition concentrations are within physiological range suggests that cation-mediated conformational changes of SP-A could be operative in vivo.

Human proteolipid protein (PLP) mediates winding and adhesion of phospholipid membranes but prevents their fusion.

Proteolipid protein (PLP or lipophilin) is a highly conserved, strongly hydrophobic, integral membrane protein, and is the major protein component of central nervous system myelin. Although PLP has been implicated in many functions, its in vivo role is still uncertain. Here, we report the investigation of PLP's putative adhesive function using purified PLP and reconstituted phospholipid vesicles made of either 100% phosphatidylcholine (PC), or a mixture of 92% PC and 8% phosphatidylserine (PS), by weight. PLP-induced changes in the phospholipid bilayer surfaces were directly examined by transmission electron microscopy. We found that upon the introduction of PLP, larger lipid vesicles became smaller and unilamellar. At the PLP:lipid molar ratio of 1:20, vesicle membranes rolled onto themselves forming 'croissant'-like structures that subsequently adhered to each other. The phenomena of PLP-induced bilayer rolling and adhesion were dependent on the concentration of PLP and the period of incubation, but were independent of the presence of calcium and types of phospholipids (PC or PC:PS). Furthermore, the presence of PLP in the lipid bilayers prevented the fusion of membranes. These findings show that PLP can induce membrane 'winding' while preventing the fusion of adjacent lipid bilayers. Hence, our data provide direct evidence for PLP's suspected function of membrane adhesion, and also suggest that PLP could potentially play a role in the formation of the myelin sheath.

Structural changes of surfactant protein A induced by cations reorient the protein on lipid bilayers.

Surfactant protein A (SP-A) is an octadecameric hydrophilic glycoprotein and is the major protein component of pulmonary surfactant. This protein complex plays several roles in the body, such as regulation of surfactant secretion, recycling and adsorption of surfactant lipids, and non-serum-induced immune response. Many of SP-A's activities are dependent upon the presence of cations, especially calcium. Here, we have studied in vitro the effect of cations on the interaction of purified bovine SP-A with phospholipid vesicles made of dipalmitoylphosphatidylcholine and unsaturated phosphatidylcholine. We have found that SP-A octadecamers exist in an "opened-bouquet" conformation in the absence of cations and interact with lipid membranes via one or two globular headgroups. Calcium-induced structural changes in SP-A lead to the formation of a clearly identifiable stem in a "closed-bouquet" conformation. This change, in turn, seemingly results in all of SP-A's globular headgroups interacting with the lipid membrane surface and with the stem pointing away from the membrane surface. These results represent direct evidence that the headgroups of SP-A (comprising carbohydrate recognition domains), and not the stem (comprising the amino-terminus and collagen-like region), interact with lipid bilayers. Our data support models of tubular myelin in which the headgroups, not the tails, interact with the lipid walls of the lattice.

Surfactant protein A (SP-A) forms a novel supraquaternary structure in the form of fibers.

We have found by transmission electron microscopy that bovine surfactant protein A (SP-A) formed extended fibers in the presence of calcium. On phosphatidylcholine or especially dipalmitoylphosphatidylcholine monolayers, SP-A at roughly 0.005 mg/ml formed large numbers of fibers and elaborate fibrous networks. This observation suggested that the weak protein:protein interactions amongst free SP-As could be stabilized by phospholipids. In the presence of glycolipid GM1-ganglioside, SP-A's globular headgroup regions appeared enlarged and only small non-fibrous clusters were observed.

SFV topoisomerase: sequence specificity in a genetically mapped interval.

Poxviral DNA topoisomerases are sequence-specific enzymes whose activities are thought to influence such diverse processes as transcription, DNA replication, and genetic recombination. To obtain further insights into the relatedness of these enzymes, and their influence on virus-mediated recombination, we have determined the target-specificity and other catalytic properties of the Shope fibroma virus (SFV) topoisomerase. SFV topoisomerase was expressed in Escherichia coli and purified as a glutathione S-transferase (GST) or (his)6-tagged fusion protein. The recombinant Leporipox-virus (SFV) enzyme displayed catalytic properties very similar to vaccinia topoisomerase. In particular SFV topoisomerase recognizes the same pentanucleotide motif [5'-(C/T)CCTT-3'] and promotes the same DNA relaxation, strand transfer, and strand cleavage reactions catalyzed by the Orthopoxviral (vaccinia) enzyme. The SFV enzyme can also efficiently cleave DNA 3' of the variant site 5'-CCCTG-3' in certain sequence contexts. These studies identified several sites where SFV topoisomerases interact with a recombinational substrate and permitted a comparison of recombination frequencies across intervals which did, or did not, span these sites. We failed to detect any effect of topoisomerase-recognition sites on recombination frequencies, except for a small (< 2-fold) stimulation seen when the substrates encoded a nearby poxviral promoter. This and other work shows that poxviral topoisomerases from several genera share common target specificities, but other enzymatic systems probably catalyze the high-frequency recombination seen in poxvirus-infected cells.