Characterization of the Sesbania rostrata Phytochelatin Synthase Gene: Alternative Splicing and Function of Four Isoforms

Phytochelatins (PCs) play an important role in detoxification of heavy metals in plants. PCs are synthesized from glutathione by phytochelatin synthase (PCS), a dipeptidyltransferase. Sesbania rostrata is a tropical legume plant that can tolerate high concentrations of Cd and Zn. In this study, the S. rostrata PCS gene (SrPCS) and cDNAs were isolated and characterized. Southern blot and sequence analysis revealed that a single copy of the SrPCS gene occurs in the S. rostrata genome, and produces four different SrPCS mRNAs and proteins, SrPCS1–SrPCS4, by alternative splicing of the SrPCS pre-mRNA. The SrPCS1 and SrPCS3 proteins conferred Cd tolerance when expressed in yeast cells, whereas the SrPCS2 and SrPCS4 proteins, which lack the catalytic triad and the N-terminal domains, did not. These results suggested that SrPCS1 and SrPCS3 have potential applications in genetic engineering of plants for enhancing heavy metal tolerance and phytoremediation of contaminated soils

Automated Solid-Phase Subcloning Based on Beads Brought into Proximity by Magnetic Force

In the fields of proteomics, metabolic engineering and synthetic biology there is a need for high-throughput and reliable cloning methods to facilitate construction of expression vectors and genetic pathways. Here, we describe a new approach for solid-phase cloning in which both the vector and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for efficient subcloning in an automated manner to rapidly generate a large number of gene constructs in various vectors intended for high throughput applications

Structure-Based Analysis of Five Novel Disease-Causing Mutations in 21-Hydroxylase-Deficient Patients

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most frequent inborn error of metabolism, and accounts for 90–95% of CAH cases. The affected enzyme, P450C21, is encoded by the CYP21A2 gene, located together with a 98% nucleotide sequence identity CYP21A1P pseudogene, on chromosome 6p21.3. Even though most patients carry CYP21A1P-derived mutations, an increasing number of novel and rare mutations in disease causing alleles were found in the last years. In the present work, we describe five CYP21A2 novel mutations, p.R132C, p.149C, p.M283V, p.E431K and a frameshift g.2511_2512delGG, in four non-classical and one salt wasting patients from Argentina. All novel point mutations are located in CYP21 protein residues that are conserved throughout mammalian species, and none of them were found in control individuals. The putative pathogenic mechanisms of the novel variants were analyzed in silico. A three-dimensional CYP21 structure was generated by homology modeling and the protein design algorithm FoldX was used to calculate changes in stability of CYP21A2 protein. Our analysis revealed changes in protein stability or in the surface charge of the mutant enzymes, which could be related to the clinical manifestation found in patients

A Conformation-Dependent Epitope in Addison’s Disease and Other Endocrinological Autoimmune Diseases Maps to a Carboxyl-Terminal Functional Domain of Human Steroid 21-Hydroxylase

Abstract Idiopathic Addison’s disease develops as a consequence of autoimmune destruction of steroid-producing cells in the adrenal gland. A major autoantigen is 21-hydroxylase (21OH; P450c21), which is involved in the biosynthesis of cortisol and aldosterone in the adrenal cortex. We selected a number of functionally important 21OH amino acid substitutions, found in patients with congenital adrenal hyperplasia, to study their effects on the binding of 21OH autoantibodies (21OHAb) to 21OH. The ability of 21OHAb to bind in vitro transcribed and translated wild-type 21OH and five different 21OH mutant proteins was quantified by liquid-phase assays. Sera from 21OHAb-positive patients with idiopathic Addison’s disease (n = 24), Graves’ disease (n = 3), and insulin-dependent diabetes mellitus (n = 1) were used. While the P105L, delE196, and G291S mutations had no effect on autoantibody binding, the P453S mutation had a considerable effect, and the R483P mutation almost completely abolished binding. Synthetic peptides corresponding to linear epitopes defined by amino acids 447–461 and 477–491 were unable to compete with wild-type 21OH for binding to autoantibodies. Direct 21OH DNA sequencing could not reveal any specific genetic variation in alleles found in 21OHAb-positive patients. We conclude that the region involving R483 plays a key role in the formation of a three-dimensional epitope in a functionally important C-terminal domain of the enzyme.</jats:p

Bead-based subcloning strategy.

<p>(<b>A</b>) An insert is excised from its donor vector, and capped with a biotin-containing oligo in a one-pot reaction. The insert is then attached to a streptavidin-coated bead and ligated to an immobilized acceptor vector, which has a compatible restriction site (here NotI). A non-productive fragment of the donor vector is also attached to bead, but cannot participate in further ligation steps due to a non-compatible restriction site (AsiSI). The DNA construct is released from bead via cutting at unique restriction site (here AscI). Details are found in Supplemental Information. (<b>B</b>) Confocal microscope image of an inter-bead ligation. Acceptor-vector beads are green (Alexa647 label) and insert beads are red (Alexa488 label). The insert is ITGA2b (1 350 bp) and the acceptor vector is pLentiHAp (8 180 bp). The inter-bead distance is much shorter than that expected for an extended DNA ligation product (ligation product expected at 9 530 bp, or 3.2 µm), which suggests multiple inter-bead connections. (<b>C</b>) A three-bead ligation. The target gene and acceptor vector are the same as in (A) Here the interbead distances are larger, indicating fewer DNA connections between the beads.</p