Molecular cloning and characterization of Siamese crocodile (Crocodylus siamensis) copper, zinc superoxide dismutase (CSI-Cu,Zn-SOD) gene.

Superoxide dismutase (SOD, EC 1.15.1.1) is an antioxidant enzyme found in all living cells. It regulates oxidative stress by breaking down superoxide radicals to oxygen and hydrogen peroxide. A gene coding for Cu,Zn-SOD was cloned and characterized from Siamese crocodile (Crocodylus siamensis; CSI). The full-length expressed sequence tag (EST) of this Cu,Zn-SOD gene (designated as CSI-Cu,Zn-SOD) contained 462bp encoding a protein of 154 amino acids without signal peptides, indicated as intracellular CSI-Cu,Zn-SOD. This agreed with the results from the phylogenetic tree, which indicated that CSI-Cu,Zn-SOD belonged to the intracellular Cu,Zn-SOD. Chromosomal location determined that the CSI-Cu,Zn-SOD was localized to the proximal region of the Siamese crocodile chromosome 1p. Several highly conserved motifs, two conserved signature sequences (GFHVHEFGDNT and GNAGGRLACGVI), and conserved amino acid residues for binding copper and zinc (His(47), His(49), His(64), His(72), His(81), Asp(84), and His(120)) were also identified in CSI-Cu,Zn-SOD. Real-time PCR analysis showed that CSI-Cu,Zn-SOD mRNA was expressed in all the tissues examined (liver, pancreas, lung, kidney, heart, and whole blood), which suggests a constitutively expressed gene in these tissues. Expression of the gene in Escherichia coli cells followed by purification yielded a recombinant CSI-Cu,Zn-SOD, with Km and Vmax values of 6.075mM xanthine and 1.4×10(-3)mmolmin(-1)mg(-1), respectively. This Vmax value was 40 times lower than native Cu,Zn-SOD (56×10(-3)mmolmin(-1)mg(-1)), extracted from crocodile erythrocytes. This suggests that cofactors, protein folding properties, or post-translational modifications were lost during the protein purification process, leading to a reduction in the rate of enzyme activity in bacterial expression of CSI-Cu,Zn-SOD.

Reduction in the structural instability of cloned eukaryotic tandem-repeat DNA by low-temperature culturing of host bacteria.

Summary For accurate analyses of eukaryotic tandem-repeat DNA, it is often required to clone a genomic DNA fragment into a bacterial plasmid. It is, however, a serious problem that tandem-repeat DNA is frequently subjected to structural changes during maintenance or amplification in the host bacteria. Here, we show an example of a clear difference in the instability of tandem-repeat DNA between different culturing temperatures. A fragment of monkey centromeric DNA carried by pUC19 was considerably degraded by culturing bacteria at 37 °C, but the damage was reduced at 25 °C. Thus, culturing temperature is a significant factor for avoiding degradation, in addition to the genotype of the host bacteria.

Higher-order repeat structure in alpha satellite DNA occurs in New World monkeys and is not confined to hominoids.

Centromeres usually contain large amounts of tandem repeat DNA. Alpha satellite DNA (AS) is the most abundant tandem repeat DNA found in the centromeres of simian primates. The AS of humans contains sequences organized into higher-order repeat (HOR) structures, which are tandem arrays of larger repeat units consisting of multiple basic repeat units. HOR-carrying AS also occurs in other hominoids, but results reported to date for phylogenetically more remote taxa have been negative. Here we show direct evidence for clear HOR structures in AS of the owl monkey and common marmoset. These monkeys are New World monkey species that are located phylogenetically outside of hominoids. It is currently postulated that the presence of HOR structures in AS is unique to hominoids. Our results suggest that this view must be modified. A plausible explanation is that generation of HOR structures is a general event that occurs occasionally or frequently in primate centromeres, and that, in humans, HOR-carrying AS became predominant in the central region of the centromere. It is often difficult to assemble sequence reads of tandem repeat DNAs into accurate contig sequences; our careful sequencing strategy allowed us to overcome this problem.

Expression and purification of dalcochinase, a beta-glucosidase from Dalbergia cochinchinensis Pierre, in yeast and bacterial hosts.

The coding sequence of the mature dalcochinase, a beta-glucosidase from Dalbergia cochinchinensis Pierre, was cloned and expressed in various systems. Expression in Escherichia coli resulted in an insoluble protein, which could be made soluble by co-expression with bacterial chaperonin GroESL. However, the enzyme had no activity. Recombinant expression in Pichia pastoris and Saccharomyces cerevisiae yielded an active enzyme. Dalcochinase was expressed under methanol induction in P. pastoris, since this was much more efficient than constitutive expression in P. pastoris or in S. cerevisiae. Addition of 0.5% casamino acids to the culture medium stabilized the pH of the culture and increased the protein yield by 3- to 5-folds. Insertion of a polyhistidine-tag either after the N-terminal alpha factor signal sequence or at the C-terminus failed to assist in purification by immobilized metal-ion affinity chromatography (IMAC) due to post-translational processing at both termini. A new construct of dalcochinase with an N-terminal truncation following the propeptide and eight histidine residues enabled its purification by IMAC, following hydrophobic interaction chromatography. The purified recombinant dalcochinase was apparently composed of differently post-translationally modified forms, but had kinetic properties and pH and temperature optima comparable to natural dalcochinase. The procedures reported here overcome the limitation in enzyme supply from natural sources, and allow further studies on structure-function relationships in this enzyme.