Portal fusion protein constraints on function in DNA packaging of bacteriophage T4.

Architecturally conserved viral portal dodecamers are central to capsid assembly and DNA packaging. To examine bacteriophage T4 portal functions, we constructed, expressed and assembled portal gene 20 fusion proteins. C-terminally fused (gp20-GFP, gp20-HOC) and N-terminally fused (GFP-gp20 and HOC-gp20) portal fusion proteins assembled in vivo into active phage. Phage assembled C-terminal fusion proteins were inaccessible to trypsin whereas assembled N-terminal fusions were accessible to trypsin, consistent with locations inside and outside the capsid respectively. Both N- and C-terminal fusions required coassembly into portals with approximately 50% wild-type (WT) or near WT-sized 20am truncated portal proteins to yield active phage. Trypsin digestion of HOC-gp20 portal fusion phage showed comparable protection of the HOC and gp20 portions of the proteolysed HOC-gp20 fusion, suggesting both proteins occupy protected capsid positions, at both the portal and the proximal HOC capsid-binding sites. The external portal location of the HOC portion of the HOC-gp20 fusion phage was confirmed by anti-HOC immuno-gold labelling studies that showed a gold 'necklace' around the phage capsid portal. Analysis of HOC-gp20-containing proheads showed increased HOC protein protection from trypsin degradation only after prohead expansion, indicating incorporation of HOC-gp20 portal fusion protein to protective proximal HOC-binding sites following this maturation. These proheads also showed no DNA packaging defect in vitro as compared with WT. Retention of function of phage and prohead portals with bulky internal (C-terminal) and external (N-terminal) fusion protein extensions, particularly of apparently capsid tethered portals, challenges the portal rotation requirement of some hypothetical DNA packaging mechanisms.

Selective anterior fusion of thoracolumbar/lumbar curves in adolescents: when can the associated thoracic curve be left unfused?

STUDY DESIGN: A retrospective multicenter study was conducted to investigate patients with a major thoracolumbar/lumbar adolescent idiopathic scoliosis and an associated minor thoracic curve treated with an anterior instrumentation and fusion of the lower curve. OBJECTIVE: To establish criteria for determining when such curves can be successfully treated by an anterior only procedure of the lower curve with acceptable spinal balance and residual thoracic curve. SUMMARY OF BACKGROUND DATA: Anterior spinal instrumentation techniques have been proved effective for the management of isolated thoracolumbar/lumbar scoliosis with small compensatory thoracic curves. The success of a selective anterior fusion when the associated thoracic curve had some structural changes in a small preliminary study was variable and was the stimulus for this study. METHODS: A multicenter study involved 49 adolescent patients with a major thoracolumbar/lumbar curve in which the associated minor thoracic curve measured between 30 degrees and 55 degrees. In all the patients, the apical vertebra of the lower curve lay outside the midsacral line, and the thoracic apical vertebra fell outside a line dropped from the center of C7. Multiple radiographic parameters were evaluated. The Risser sign, height, weight, onset of menses, and closure of the triradiate cartilages were studied to access the patients' maturity. All the patients were observed at least 2 years. Patients were considered to have a satisfactory result if the thoracic curve at the final follow-up assessment measured 40 degrees or less, if balance and sagittal alignment were reasonable, and if additional procedures were not required. RESULTS: At final follow-up assessment, two groups emerged. Group 1 (n = 43) had satisfactory results. The preoperative thoracic curve in this group averaged 40 degrees and 26 degrees after surgery. The lumbar curve averaged 56 degrees before surgery and 22 degrees after surgery. Group 2 (n = 6) had unsatisfactory results. The average thoracic curve was 49 degrees before surgery 54 degrees after surgery, whereas the lumbar curve averaged 59 degrees before surgery and 27 degrees after surgery. Three of these patients underwent posterior thoracic instrumentation and fusion. CONCLUSIONS: Statistical analysis showed that a successful surgical outcome was dependent on both the structural changes in the thoracic curve and the patient's maturity. The thoracolumbar/lumbar-thoracic (TL/L:T) Cobb ratio in combination with the degree of the thoracic curve on lateral bending was the best predictor among the structural indexes. Of 44 patients with a TL/L:T Cobb ratio of 1.25 or greater and/or a thoracic curve, which bent out to 20 degrees or less, 42 had a satisfactory result. The best predictor among the maturity indexes was closure of the triradiate cartilages. Of 43 patients in whom the triradiate cartilages were closed, 42 had satisfactory results. When this data is combined, the outcome for the thoracic curve can be reasonably predicted.

Isolation and characterization of T4 bacteriophage gp17 terminase, a large subunit multimer with enhanced ATPase activity.

Phage T4 terminase is a two-subunit enzyme that binds to the prohead portal protein and cuts and packages a headful of concatameric DNA. To characterize the T4 terminase large subunit, gp17 (70 kDa), gene 17 was cloned and expressed as a chitin-binding fusion protein. Following cleavage and release of gp17 from chitin, two additional column steps completed purification. The purification yielded (i) homogeneous soluble gp17 highly active in in vitro DNA packaging ( approximately 10% efficiency, >10(8) phage/ml of extract); (ii) gp17 lacking endonuclease and contaminating protease activities; and (iii) a DNA-independent ATPase activity stimulated >100-fold by the terminase small subunit, gp16 (18 kDa), and modestly by portal gp20 and single-stranded binding protein gp32 multimers. Analyses revealed a preparation of highly active and slightly active gp17 forms, and the latter could be removed by immunoprecipitation using antiserum raised against a denatured form of the gp17 protein, leaving a terminase with the increased specific activity (approximately 400 ATPs/gp17 monomer/min) required for DNA packaging. Analysis of gp17 complexes separated from gp16 on glycerol gradients showed that a prolonged enhanced ATPase activity persisted after exposure to gp16, suggesting that constant interaction of the two proteins may not be required during packaging.