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 new locus for autosomal recessive hypercholesterolemia maps to human chromosome 15q25-q26

High serum cholesterol is an established risk factor for cardiovascular disease and is the prime target for therapeutic intervention in large groups of patients. The development of modern treatments for this major risk factor was propelled by the early realization that forms of severe hypercholesterolemia could be caused by dominantly inherited defects in the LDL receptor or in the APOB gene. Further understanding of the mechanisms contributing to early atherosclerosis will allow for new targets for therapy. We therefore identified and investigated the genetics of families from Sardinia that have recessive inheritance of precocious hypercholesterolemia. We used five families in an analysis of linkage of the autosomal recessive hypercholesterolemia locus, termed "ARH1," to chromosome 15q25-q26. A genomewide search mapped the disease-causing gene with a LOD score of 3.3 and excluded major contributions to the phenotype of other genes. A candidate gene present in the mapped chromosome region-the ligand-activated liver-transcription-factor gene ARP1 (apolipoprotein regulatory-protein gene)-has been excluded after DNA sequencing. The close-bred nature of the Sardinian population offers unique opportunities for isolation of this hypercholesterolemia-causing gene

The presence of magnet increases the extent of ligation in bead-bead subcloning.

<p>Flow cytometry of fluorescent-labeled beads allows quantification of bead-bead ligations. Insert beads are red-labeled (Alexa488 label) and acceptor-vector beads are green-labeled (Alexa647 label). Successful bead-bead ligations appear at high FL-1 and FL-6 readouts (boxed). The insert is ITGA2b (1 350 bp) and the acceptor vector is pLenti1 (8 180 bp). (A) Ligation in the absence of magnet. Approximately 0.5% of acceptor beads are ligated. (B) Ligation in the presence of magnet. Approximately 7% of the acceptor beads are ligated, a 14-fold increase in extent of ligation.</p

Statistics of automated bead-bead subcloning of 95 target genes.

<p>Statistics of automated bead-bead subcloning of 95 target genes.</p

Ligation efficiencies of bead-based subcloning strategies.^*

*<p> <i>Vector and donor beads were loaded at 0.5 ng DNA/µg bead.</i></p

Ligation of solution phase DNA to a bead-immobilized vector.

<p>(<b>A</b>) A fluorescence-based assay for determining extent of ligation. Beads with immobilized vector are incubated with a Alexa 488 fluorescent oligo. The extent of ligation is measured via flow cytometry. Bead loading was at 1 ng vector DNA (pHISZ)/ug bead vector. Positive: Beads in which pHISZ vector is fully fluorescently labeled. Ligation: Beads after ligation. Negative: Beads in which pHISZ vector is not fluorescently labeled. The extent of ligation f is measured as a percentage of the Positive signal. (<b>B</b>) Extent of ligation is reduced at high bead loadings.</p