A high efficiency method for site-directed mutagenesis with any plasmid.

A procedure is described which allows for the site-directed mutagenesis of DNA segments in any double-stranded plasmid with high efficiency. There are no limitations as to the position of the mutation. The protocol involves only simple enzymatic manipulations and no difficult to control operations, such as partial digestions, are required. The method was developed and used to mutagenize two different genes (encoding human interferon-beta and interleukin-2) cloned in a eukaryotic expression vector. For ten mutageneses with different oligodeoxyribonucleotides the average yield of mutants was 60%.

The influence of pulmonary artery banding on outcome after the Fontan operation.

Thirty-eight patients were selected from a total of 120 patients who underwent the Fontan operation between 1974 and 1988. They were classified into two groups. Group 1 consisted of 18 patients with previous pulmonary artery banding at a mean age of 7 months (2 days to 59 months), and group 2 comprised 20 patients with native pulmonary stenosis. In group 1, 10 children had tricuspid atresia (seven with normally connected and three with transposed great arteries), six had double-inlet ventricle, and two had complex heart malformations. Group 2 consisted of 12 patients with tricuspid atresia and normally connected great arteries, six with double-inlet ventricle, and two with complex malformations. The following clinical and hemodynamic parameters at cardiac catheterization and cineangiocardiography were determined in both groups before the Fontan operation: age and body surface area, hemoglobin concentration and hematocrit value, atrial and pulmonary artery pressures, end-diastolic pressure of the systemic ventricle, arterial oxygen saturation, pulmonary/systemic flow ratio, end-diastolic volume, ejection fraction and mass of the systemic ventricle, cardiac index, and Nakata index. After the Fontan operation in all patients, the presence or absence of pericardial and pleural effusions, ascites, protein-losing enteropathy, and liver and kidney dysfunction was assessed and the clinical status was classified according to New York Heart Association criteria. All preoperative and postoperative parameters were tested for differences between the two groups, and they were compared with normal values. Hematocrit value was higher in group 2 than in group 1 (57.8% versus 53.1%; p less than 0.05). Ventricular mass index was increased in group 1 when compared with group 2 (125.8 gm/m2 versus 87 gm/m2; p less than 0.05). Severe pericardial effusions in the early postoperative period were significantly more frequent in group 1 and were particularly prevalent in the subgroup with long-standing pulmonary artery banding (p less than 0.01). Subaortic stenosis was observed more frequently in group 1. The remaining parameters were not statistically different between the two groups. We conclude that the significant increment in ventricular mass after pulmonary artery banding may represent a risk for unfavorable outcome after the Fontan operation, which increases with time. Therefore, long-standing pulmonary artery banding as a palliative procedure for candidates for the Fontan operation should be avoided.