Incidental findings in CT imaging of coronary artery bypass grafts: results from a Canadian multicenter prospective cohort.

OBJECTIVE: To assess the prevalence and clinical significance of incidental findings identified during computed tomography imaging of coronary artery bypass grafts. RESULTS: This prospective study includes 144 patients undergoing coronary graft patency assessment using computed tomography. Incidental findings were classified as significant if they were considered to need an immediate action or treatment, short-term work-up or follow-up, or minor. A total of 211 incidental findings were present in 109 (75.7%) patients. Seventy-one incidental findings (33.6%) were cardiac and 140 (66.4%) were extracardiac. Most common cardiac incidental findings were atrial dilatation [39 patients, 48 incidental findings (67.6%)] and aortic valve calcifications (7 patients, 9.9%). Among the 140 extracardiac incidental findings, the most common were lung nodules (51 patients, 54 nodules, 38.6%), and emphysema (21 patients, 15%). Thirty-six (25.7%) extracardiac incidental findings were significant and notably, 23 (63.9%) were lung nodules. Follow-up was recommended in 37 cases, among which all patients with significant lung nodules (23 patients, 62.2%). In conclusion, most common computed tomography incidental findings in patients with coronary grafts were lung nodules and emphysema.

A cardioplegia circuit with versatility: the 'ReVerse' system. How to do it.

Various methods of cardioplegia administration have been used in cardiac surgery: crystalloid, blood and mixed crystalloid/blood. Each of these types of cardioplegia administration typically needs a different circuit. This may correspond to an increase in cost and the time needed to change the circuit if required. When various modifications are performed on the circuit, this also increases the risk of contamination. In order to simplify the management of differing cardioplegia circuits, we devised one circuit for all solutions in all situations by adding one modification. The ReVerse cardioplegia circuit system is a description of a two-pump cardioplegia circuit which is adaptable to either blood or crystalloid cardioplegia. The change from one mode to another requires a manoeuvre of two clamps, allowing the blood solution to travel through shunt tubing into the apposite pumphead. In our experience the versatility of this circuit is a fast, safe method to administrate all types of cardioplegia solution, saving the space taken up by storing multiple circuits.

SCP1, a 356,023 bp linear plasmid adapted to the ecology and developmental biology of its host, Streptomyces coelicolor A3(2).

The sequencing of the entire genetic complement of Streptomyces coelicolor A3(2) has been completed with the determination of the 365,023 bp sequence of the linear plasmid SCP1. Remarkably, the functional distribution of SCP1 genes somewhat resembles that of the chromosome: predicted gene products/functions include ECF sigma factors, antibiotic biosynthesis, a gamma-butyrolactone signalling system, members of the actinomycete-specific Wbl class of regulatory proteins and 14 secreted proteins. Some of these genes are among the 18 that contain a TTA codon, making them targets for the developmentally important tRNA encoded by the bldA gene. RNA analysis and gene fusions showed that one of the TTA-containing genes is part of a large bldA-dependent operon, the gene products of which include three proteins isolated from the spore surface by detergent washing (SapC, D and E), and several probable metabolic enzymes. SCP1 shows much evidence of recombinational interactions with other replicons and transposable elements during its history. For example, it has two sets of partitioning genes (which may explain why an integrated copy of SCP1 partially suppressed the defective partitioning of a parAB-deleted chromosome during sporulation). SCP1 carries a cluster of probable transfer determinants and genes encoding likely DNA polymerase III subunits, but it lacks an obvious candidate gene for the terminal protein associated with its ends. This may be related to atypical features of its end sequences.

Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2).

Streptomyces coelicolor is a representative of the group of soil-dwelling, filamentous bacteria responsible for producing most natural antibiotics used in human and veterinary medicine. Here we report the 8,667,507 base pair linear chromosome of this organism, containing the largest number of genes so far discovered in a bacterium. The 7,825 predicted genes include more than 20 clusters coding for known or predicted secondary metabolites. The genome contains an unprecedented proportion of regulatory genes, predominantly those likely to be involved in responses to external stimuli and stresses, and many duplicated gene sets that may represent 'tissue-specific' isoforms operating in different phases of colonial development, a unique situation for a bacterium. An ancient synteny was revealed between the central 'core' of the chromosome and the whole chromosome of pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae. The genome sequence will greatly increase our understanding of microbial life in the soil as well as aiding the generation of new drug candidates by genetic engineering.

Endothelin receptor blockade improves endothelial function in human internal mammary arteries.

OBJECTIVE: Endothelial dysfunction, specifically endothelium-derived contracting factors have been implicated in the development of arterial conduit vasospasm. The potent vasoconstrictor endothelin-1 (ET-1) has received much attention in this regard. The present study was designed to evaluate the role of ET-1 in the development of endothelial dysfunction in human internal mammary arteries (IMA). To this aim, we examined the effects of specific and non-specific ET-receptor antagonists on endothelial function (assessed using acetylcholine (ACh)-induced vasodilation) in segments of IMA obtained during coronary artery bypass graft (CABG) surgery. METHODS: Vascular segments of IMA were obtained from 51 patients undergoing elective coronary artery bypass graft (CABG) surgery and in vitro endothelium-dependent and -independent responses to ACh and sodium nitroprusside (SNP) were assessed. Isometric dose response curves (DRC) to ACh and SNP were constructed in pre-contracted rings in the presence and absence of bosentan (ET(A/B) receptor antagonist, 3 microM), BQ-123 (ET(A) antagonist, 1 microM) and BQ-788 (ET(B) antagonist, 1 microM) using the isolated organ bath apparatus. Percent maximum relaxation (%E(max)) and sensitivity (pEC(50)) were compared between interventions. RESULTS: ACh caused dose-dependent endothelium-mediated relaxation in IMA (%E(max) 43+/-4, pEC(50) 6. 74+/-0.12). In the presence of bosentan, BQ-123 and BQ-788 ACh-induced relaxation was significantly augmented (%E(max) bosentan 60+/-3, BQ-123 56+/-4, BQ-788 53+/-5 vs. control 43+/-4, P<0.05) without affecting sensitivity. The effects of these antagonists were endothelium-specific since endothelium-independent responses to SNP remained unaltered. Furthermore, the beneficial effects were independently and maximally mediated by ET(A) and ET(B) receptors (%E(max) BQ-123 56+/-4 vs. BQ-788 53+/-5 vs. bosentan 60+/-3, P>0. 05). CONCLUSIONS: These data uncover, for the first time, beneficial effects of ET receptor blockade on endothelial-dependent vasorelaxation in human IMA.

The ram-dependence of Streptomyces lividans differentiation is bypassed by copper.

The onset of morphological differentiation in Streptomyces lividans is intrinsically delayed in comparison to Streptomyces coelicolor, but can be advanced by adding extra copper to the medium. Copper-specific chelators block aerial hyphae formation in both strains illustrating the crucial role of copper in morphogenesis. The S. coelicolor ram cluster was isolated as a clone that complements the copper-dependent differentiation of S. lividans. The S. lividans ram cluster was cloned and shown to be 99.6% identical to the S. coelicolor clone. The difference in development between S. lividans and S. coelicolor could neither be related to functional differences between the two ram clusters nor to differences in the transcription level. In both strains the low level of ramAB transcription correlated with aerial mycelium formation and was coupled to the upstream ORF ramS. An increased ramAB expression level in S. lividans by the introduction of an extra copy of ram stimulated the development. In S. lividans disruption of ramABR resulted in the inability to produce aerial hyphae. Conversely, the identical mutant of S. coelicolor retained its developmental capacities, indicating the presence of a ram-independent developmental route that is not present or not activated in S. lividans. Aerial hyphae and spore formation in the S. lividans ramABR mutant was restored when grown near wild-type strains, suggesting that the ram gene products are involved in transport of a factor essential for normal development. In addition, an elevated copper concentration in the medium also relieved the developmental block of these mutants. These findings suggest that higher copper concentrations render this ram-associated factor obsolete.

Tetrahydrobiopterin improves endothelial function in human saphenous veins.

OBJECTIVES: Diminished production of nitric oxide has been linked to saphenous vein endothelial dysfunction. Tetrahydrobiopterin is an obligate cofactor for the oxidation of L -arginine by nitric oxide synthase in the production of nitric oxide by endothelial cells. The objective of the present study was to examine whether the exogenous addition of tetrahydrobiopterin improves endothelial function in saphenous veins from patients undergoing coronary artery bypass graft operations. METHODS: Vascular segments of saphenous veins were obtained from 17 patients undergoing elective coronary artery bypass grafting, and in vitro endothelium-dependent and endothelium-independent responses to acetylcholine and sodium nitroprusside were assessed. Isometric dose-response curves were constructed in precontracted rings in the presence and absence of tetrahydrobiopterin (0.1 mmol/L) with the use of the organ bath apparatus. The percentages of maximum relaxation and sensitivity were compared between interventions. RESULTS: Acetylcholine caused dose-dependent endothelium-mediated relaxation in saphenous veins. In the presence of tetrahydrobiopterin, acetylcholine-induced relaxation was significantly augmented (percentage maximum relaxation, 16.8% +/- 2.9% vs control 7.5% +/- 1.8%; P =.003) without an effect on agonist sensitivity. These effects were endothelium-specific because endothelium-independent responses to sodium nitroprusside were preserved. CONCLUSIONS: These data uncover beneficial effects of acute tetrahydrobiopterin addition on endothelial function in human vessels. Because endothelial dysfunction has been implicated in the development of graft failure, studies aimed at chronic delivery of tetrahydrobiopterin would be useful in determining the contribution of this cofactor toward saphenous vein atherosclerosis.

Postinspiratory activity of costal and crural diaphragm.

Because the first stage of expiration or "postinspiration" is an active neurorespiratory event, we expect some persistence of diaphragm electromyogram (EMG) after the cessation of inspiratory airflow, as postinspiratory inspiratory activity (PIIA). The costal and crural segments of the mammalian diaphragm have different mechanical and proprioceptive characteristics, so postinspiratory activity of these two portions may be different. In six canines, we implanted chronically EMG electrodes and sonomicrometer transducers and then sampled EMG activity and length of costal and crural diaphragm segments at 4 kHz, 10.2 days after implantation during wakeful, resting breathing. Costal and crural EMG were reviewed on-screen, and duration of PIIA was calculated for each breath. Crural PIIA was present in nearly every breath, with mean duration 16% of expiratory time, compared with costal PIIA with duration -2. 6% of expiratory time (P < 0.002). A linear regression model of crural centroid frequency vs. length, which was computed during the active shortening of inspiration, did not accurately predict crural EMG centroid frequency values at equivalent length during the controlled relaxation of postinspiration. This difference in activation of crural diaphragm in inspiration and postinspiration is consistent with a different pattern of motor unit recruitment during PIIA.

[Over-expression of a polyketide synthase (PKS) module of a giant polyene antibiotic gene cluster in E. coli by double induction].

A 2,671 bp DNA carrying a type I PKS module with KS and AT domains from Streptomyces sp. FR-008 was cloned in-frame into the BamHI site immediately downstream of the PT7 promoter of the E. coli expression vector pET-15b, no considerable expression under IPTG induction was detected. The same PKS gene cloned downstream of the tandem PRPL promoters of pBV220 also yielded no over-expression under 42 degrees C induction. This gene was, however, over-expressed when it was cloned downstream of the tandem PRPT7 or PRPLPT7 promoters. In the case of the tandem PRPLPT7 promoters, the over-expression was dependent on the 42 degrees C plus IPTG double induction. While in the case of the tandem PRPT7 promoters, over-expression could be achieved when the gene was induced by IPTG or 42 degrees C individually or by IPTG and 42 degrees C double induction. Based on these experiences an expression vector pHZ330 containing the tandem PRPT7 promoters was constructed. In addition, the PKS protein expressed in E. coli was injected into rabbits to generate PKS-specific antibodies. Western blotting experiment indicated that these antibodies were PKS-specific which could be used either for the study of the PKS gene cluster or for the detection of the heterologous expression of Streptomyces sp. FR-008 PKS genes.

Eliminating ultrasonic interference from respiratory muscle EMG.

Fine wire recordings of the respiratory muscle electromyogram are often employed to represent muscle activity, and recently ultrasound-sonomicrometry has become a common method of measuring length of respiratory muscles in both acute and chronic preparations. Although recording both EMG and sonomicrometry simultaneously has become standard practice, there has not been any consideration of the potential confounding influence of ultrasound noise upon the recorded EMG spectrum. Activation of the sonomicrometry-ultrasound tranducer introduces a high frequency, high amplitude voltage pulse plus harmonics, which can contaminate the EMG spectrum directly, as well as through aliasing when EMG is sampled directly digitally. We describe the use of a new, combined, wing stabilized sonomicrometry- and EMG measurement transducer to characterize exactly the influence of ultrasound upon the crural diaphragm EMG spectrum, and the development of digital filtering techniques which effectively eliminate the ultrasound interference. Two alternative methods of avoiding ultrasound-EMG interference are also considered. The isolation and elimination of ultrasound-sonomicrometry signal interference may be important in studies where EMG and length are measured together.

Diaphragm function during sighs in awake dogs after laparotomy.

Pulmonary complications after upper abdominal surgery are usually ascribed to temporary postoperative impairment of diaphragm function, which may not originate from intrinsic, structural injury but from reflex inhibition of diaphragm contractility. Spontaneous breathing is interrupted periodically by sighs, even after upper abdominal surgery. If postoperative dysfunction of the diaphragm arises from a reflexic inhibition, then the sigh should temporarily override the inhibition and restore normal diaphragm function. We implanted sonomicrometer and electromyogram transducers chronically in six dogs by laparotomy, then directly measured length, shortening, and electromyogram activity of costal and crural diaphragm segments, parasternal intercostal, and transversus abdominis muscles an average of 8.7 (range, 1-16) d later during resting tidal breathing and sighs. In each animal we analyzed a sequence of breaths, including a sigh, when costal or crural diaphragm contractility was abnormal. With each sigh, the shape and amplitude of costal and crural diaphragm segmental shortening improved abruptly, from 0.9 and 1.4% of baseline length (% LBL) during resting breathing to 12.1 and 11.1% LBL, respectively, during sighs. The sighs were compared to CO2-stimulated breaths of equivalent tidal volume, which did not show either pattern or amplitude of shortening equivalent to sighs. We conclude that diaphragm dysfunction after laparotomy arises from a reflex inhibition, which is overridden abruptly to return diaphragm function briefly to normal during each spontaneous sigh.

Effect of dual-chamber pacing with automatic rate-drop sensing on recurrent neurally mediated syncope.

We tested the hypotheses that a dual-chamber pacemaker that paces when intrinsic rate drops abruptly would reduce the number of syncopal spells and improve the quality of life in patients with highly recurrent neurally mediated syncope. Twelve patients with highly frequent neurally mediated syncope and at least 1 syncopal spell after tilt testing received dual-chamber pacemakers with automatic rate-drop sensing. The pacemakers were implanted 17+/-26 months after tilt testing, and the patients then were followed for 12+/-2 months. We compared the time to the first recurrence of syncope, syncope frequency, and quality of life for the 2 periods between tilt testing and pacemaker implantation, and between implantation and last follow-up. Only 6 of 12 patients fainted after pacemaker insertion. The median time to syncope recurrence before and after pacing was 7 days and 5.3 months, respectively. The geometric mean frequency of faints before and after pacing was 5.0 spells/month (95% confidence interval 2.7 to 9.2) and 0.30 spells/month (95% confidence interval 0.2 to 0.4), p <0.001. After 6 months the mean perception of health on the 100-point EuroQol scale rose from 55 to 82 (p = 0.003), and the general health perception on the SF-36 scale rose from 51 to 72 (p = 0.005). Permanent dual-chamber pacing with automatic rate-drop sensing in patients with highly frequent syncope is associated with a marked reduction in the likelihood of syncope and a marked improvement in quality of life.

Government, university, and community collaboration to develop a cardiac surgery database and report of morbidity and mortality while waiting for open heart surgery.

The Provincial Advisory Committee on Cardiovascular Services was established in January of 1990 to advise concerning these services. One of the first tasks assigned was to monitor waiting times for cardiac surgery. Referring cardiologists categorized their patients into four priorities: emergency, urgent-inpatient, urgent-outpatient, and planned. Data of the southern Alberta centers for the past three years were analyzed for events while waiting for surgery. (Median time to event in days) M1=Myocardial Infarction EM=Emergency D=Day A hierarchy was used to assign the single most serious event for patients having more than one event: death>MI>readmission or change from urgent-inpatient to emergency. Events were frequent and unpredictable, particularly in outpatients. Categorization of patient suitable to wait at home for cardiac surgery is imperfect. The risk of having an event while on the waiting list is much greater for out-patients than in-patients: 12.8% (169/1323) versus 1.9% (19/1002). All adverse events for the in-patients occurred at four days--one day less than the proposed maximum waiting time. In the out-patient population, the median waiting time to experiencing adverse events ranged between 32 and 54 days. Target waiting times set by PACCS for these two categories is 14 and 56 days respectively. Total adverse events occurred to 8% of the patients on the waiting list. Median waiting time to experiencing an adverse event while on the list occurs much earlier than suspected: four days in urgent in-patients and 36 days for out-patients; well below the intended maximum of 56 days. This database proved invaluable for this important critical data collection. It is hoped it will serve as a model for similar future projects.

Right atrial and right ventricular transmural pressures in dogs and humans. Effects of the pericardium.

BACKGROUND: To determine the transmural pressure-dimension relations of the right atrium (RA) and right ventricle (RV) before and after pericardiectomy, six open-chest dogs were instrumented with pericardial balloons placed over the RA and RV free walls. METHODS AND RESULTS: PA appendage dimensions and RV free-wall segment lengths were measured using sonomicrometry. Intact-pericardium RA and RV transmural pressures were calculated by subtracting the pericardial pressures (measured using balloons) from the cavitary pressures. Pooled data from six animals with pericardium intact indicate that at RA and RV cavitary pressures of 5, 10, and 15 mm Hg, RV pericardial pressure was 4.3 +/- 0.3, 8.6 +/- 1.0, and 13.3 +/- 1.5 mm Hg, respectively, and RA pericardial pressure was 4.8 +/- 0.3, 9.6 +/- 0.6, and 14.6 +/- 0.6 mm Hg, respectively (mean +/- SD). With calculated unstressed dimensions, the cavity dimension data were normalized to strain (in percent). We determined that in the dog, RV strain would increase by 14% and RA by 68% to maintain cavitary pressure at 10 mm Hg on pericardiectomy. To compare these results with clinical data, RV (n = 7) and RA (n = 6) transmural pressures were measured using balloons in patients (age, 19 to 76 years) undergoing cardiac surgery. RA transmural pressure of six patients was 1.0 +/- 1.5 mm Hg when central venous pressures (CVPs) ranged from 3 to 16 mm Hg. RV transmural pressure equaled 1.2 +/- 1.9, 2.3 +/- 1.9, and 3.4 +/- 2.0 mm Hg when CVP was 5, 10, and 15 mm Hg, respectively. CONCLUSIONS: Pericardial constraint (as evaluated by the ratio of pericardial to intracavitary pressures when CVP is 10 mm Hg) accounted for 96% of RA cavitary pressure in the dog and 89% in humans and at least 86% of RV cavitary pressure in the dog and 77% in humans.

High-frequency homologous plasmid-plasmid recombination coupled with conjugation of plasmid SCP2* in Streptomyces.

Non-transmissible derivatives of the Streptomyces multi-copy plasmid plJ101 were mobilized, by cointegrate formation, at frequencies approaching 100% (measured per recipient) by derivatives of the conjugative, low-copy-number Streptomyces coelicolor A3(2) plasmid SCP2*. Efficient co-integrate formation required that the plasmids shared at least 112 bp sequence identity, and it occurred only during conjugation. An SCP2* plasmid gene is involved in the process. Co-integrates were presumably formed in the donor cells and transported to the recipient cells. This is a new phenomenon, not known in other bacteria.

Repeated polyketide synthase modules involved in the biosynthesis of a heptaene macrolide by Streptomyces sp. FR-008.

Genes for biosynthesis of a Streptomyces sp. FR-008 heptaene macrolide antibiotic with antifungal and mosquito larvicidal activity were cloned in Escherichia coli using heterologous DNA probes. The cloned genes were implicated in heptaene biosynthesis by gene replacement. The FR-008 antibiotic contains a 38-membered, polyketide-derived macrolide ring. Southern hybridization using probes encoding domains of the type I modular erythromycin polyketide synthase (PKS) showed that the Streptomyces sp. FR-008 PKS gene cluster contains repeated sequences spanning c. 105kb of contiguous DNA; assuming c. 5 kb for each PKS module, this is in striking agreement with the expectation for the 21-step condensation process required for synthesis of the FR-008 carbon chain. The methods developed for transformation and gene replacement in Streptomyces sp. FR-008 make it possible to genetically manipulate polyene macrolide production, and may later lead to the biosynthesis of novel polyene macrolides.

Respiratory muscle compensation for unilateral or bilateral hemidiaphragm paralysis in awake canines.

In humans and some animals, the surviving respiratory muscles are able to compensate fully for unilateral, and partially for bilateral, hemidiaphragm paralysis. To examine differential activity of individual respiratory muscles after unilateral or bilateral diaphragm paralysis, length and electromyogram (EMG) of left costal and crural diaphragm segments, parasternal intercostal, and transversus abdominis were measured directly in five awake canines after implantation with sonomicrometry transducers and bipolar EMG electrodes under three conditions: during normal breathing (NOFRZ), after infusion of local anesthetic (bupivacaine) through a cervical phrenic nerve cuff to induce reversible contralateral hemidiaphragm (CNFRZ), and after bilateral diaphragm (BIFRZ) paralysis. From NOFRZ to CNFRZ, costal, crural, parasternal, and transversus abdominis increased shortening and EMG activity to compensate for contralateral diaphragm paralysis, but the increase in activity was not equivalent for each muscle. With BIFRZ, parasternal and transversus abdominis showed further increases in activity, coordinated between both inspiration and expiration. Normalized intrabreath profiles revealed dynamic differences in development of muscle activity within each breath as paralysis worsened. Review of simultaneous muscle activities showed coordinated interactions among the compensating muscles: passive shortening of transversus, and lengthening of costal and crural, coincided with increased active inspiratory shortening of parasternal. We conclude that an integrated strategy of respiratory muscle compensation for unilateral or bilateral diaphragm paralysis occurs among chest wall, abdominal, and diaphragm segmental muscles, with relative contributions of individual muscles adjusted according to the degree of diaphragm dysfunction.

Streptomyces lividans 66 contains a gene for phage resistance which is similar to the phage lambda ea59 endonuclease gene.

The DNA of wild-type Streptomyces lividans 66 is degraded during electrophoresis in buffers containing traces of ferrous iron. S. lividans ZX1, a mutant selected for resistance to DNA degradation, simultaneously became sensitive to phi HAU3, a wide-host-range temperate bacteriophage. A DNA fragment conferring phi HAU3 resistance was cloned; it contains a phage resistance gene whose deduced amino acid sequence is similar to the phage lambda Ea59 endonuclease. The S. lividans phi HAU3 resistance does not seem to be a classical restriction-modification system, because no host-modified phages able to propagate on the wild-type strain could be isolated. The cloned fragment did not make the host DNA prone to degradation during electrophoresis, indicating that the two phenotypes are controlled by different genes which were deleted together from the chromosome of ZX1.

Respiratory muscle function during emesis in awake canines.

Emesis requires a coordinated differential recruitment of gastrointestinal smooth muscle, upper airway muscles, and several muscles involved in respiration. In seven awake intact canines we measured the electrical activity (electromyogram) and shortening of costal and crural diaphragm segments, parasternal intercostal, and transversus abdominis during emesis that was induced by instillation of apomorphine into the lower conjunctival fornix. The process of emesis was tightly coordinated with ventilation and showed four respiratory phases: baseline ventilation (Base), initial preemetic hyperventilation (Hyperv), prodromal ventilation associated with salivation and probable nausea (Prodrome), and finally retching and expulsion (Expel) of gastric contents. Ventilation was suppressed during expulsive events, but a small inspiratory airflow was interjected between expulsions. Resting electromyogram of all four muscles increased during the process of emesis, with costal and crural segments showing a marked decrease in resting length through Prodrome and Expel. To produce an expulsive maneuver, both inspiratory and expiratory muscles were activated synchronously, unlike their usual sequential activation during ventilation, with costal and crural segments and transversus abdominis showing the most shortening. The crural segment showed a biphasic length change with initial shortening and then lengthening to assist esophageal sphincter function during Expel. These results indicate a strong coordinated interaction between brain stem centers responsible for control of respiration and of emesis.

Transposition of IS117, the 2.5 kb Streptomyces coelicolor A3(2) 'minicircle': roles of open reading frames and origin of tandem insertions.

IS117 is a 2527 bp transposable element from Streptomyces coelicolor A3(2) with a circular transposition intermediate. Disruption of ORF1 of IS117, presumed to encode a transposase, abolished transposition. Deletion or mutation of ORF2 and ORF3, which overlap each other on opposite strands of IS117, caused a c. 20-fold reduction in integration frequency of the circular form of IS117 into the Streptomyces lividans chromosome or into the preferred chromosomal target site cloned on a plasmid in transformation experiments. In contrast, inactivation of ORF2/3 did not significantly influence transposition of IS117 derivatives from an already integrated state in the chromosome to the preferred target site cloned on a plasmid. ORF2 mutants apparently excised readily from the S. lividans chromosome, whereas excision of integrated wild-type IS117 derivatives to yield the unoccupied site was not detected; presumably, therefore, the circular transposition intermediate normally arises replicatively. Attempts to promote integration of a plasmid carrying the attachment site of IS117 by providing the ORF1 product in trans were unsuccessful. Most transformation of S. lividans with circular IS117 derivatives yielded tandem chromosomal insertions, which arose by co-transformation rather than dimerization of a monomeric insert. Typically, two to three transforming elements gave a transformed strain, suggesting a local concentration of transposase as a limit on integration.