ABSTRACT
A useful variant of PCR technique was devised to generate full-length Aspergillus niger arginase cDNA for expression. Briefly, a 450 bp amplicon was first constructed through overlap extension PCR (OE-PCR) by splicing in a 101 nucleotide long single-stranded megaprimer, facilitated by inclusion of an additional, shorter forward primer in the reaction. The amplicon was suitably cloned into pBlueScript to obtain pArg440 and the insert sequenced. The full-length arginase cDNA was subsequently assembled in pArg440 and moved into pET23a for heterologous expression. An interesting feature of this strategy was not to stoichiometrically incorporate the oligonucleotide megaprimer, but use it only as an early template. This OE-PCR strategy to utilize long single-stranded megaprimer may prove handy in terms of efficiency, yield and sequence choice.
Subject(s)
Arginase , Aspergillus niger/genetics , DNA, Complementary/metabolism , DNA Primers , DNA, Single-Stranded , Polymerase Chain Reaction/methods , RNA SplicingABSTRACT
The present study was planned to assess the correlation of pulmonary functions with body fat percentage in young individuals. A total of 132 (males-68, females-64) young students (18 to 21 years) with moderately sedentary lifestyle were recruited in this study. Their height, weight, body mass index (BMI) and waist to hip ratio (WHR) were measured. Pulmonary function tests (static and dynamic) were recorded on a computerized portable Schiller lung function unit SP-1. The percentage of body fat was estimated by measuring skin fold thickness at four sites (4SFT-biceps, triceps, subscapular and suprailiac) with the help of Harpenden’s caliper. In males body fat % showed negative correlation with expiratory reserve volume (ERV), forced vital capacity (FVC), maximum ventilatory volume (MVV), peak expiratory flow rate (PEFR) and forced expiratory volume at the end of first second (FEV1). It was observed that in females body fat % had negative correlation with ERV, FVC, and MVV. These results indicate that increase in percentage of body fat and central pattern of fat distribution may affect the pulmonary function t e s t s.