Nucleocapsid protein VP15 is the basic DNA binding protein of white spot syndrome virus of shrimp.

White spot syndrome virus (WSSV) is type species of the genus Whispovirus of the new family Nimaviridae. Despite the elucidation of its genomic sequence, very little is known about the virus as only 6% of its ORFs show homology to known genes. One of the structural virion proteins, VP15, is part of the nucleocapsid of the virus and shows homology to some putative baculovirus DNA binding proteins. These DNA-binding or histone-like proteins are thought to be involved in the condensation and packaging of the genome in the nucleocapsid. Using bacterially expressed VP15 fusion proteins in ELISA and Far-Western experiments showed that VP15 interacts with itself, forming homomultimers, but not with the other major structural proteins of the WSSV virion. Antibodies against phosphorylated proteins revealed that VP15 originating from different sources was not phosphorylated. WSSV VP15 binds non-specifically to double-stranded DNA, but has a clear preference to supercoiled DNA suggesting that VP15 is involved in the packaging of the WSSV genome in the nucleocapsid. This research shed further light on the composition of WSSV virions and the function of one of its nucleocapsid proteins.

Purification and characterization of juvenile hormone esterase from hemolymph of the Colorado potato beetle.

In the Colorado potato beetle (Leptinotarsa decemlineata), low juvenile hormone (JH) titers are necessary to initiate metamorphosis and diapause. Low JH titers coincide with high activities of JH esterase, which occur mainly in the hemolymph. The specific activity of JH esterase appeared to be highest in the last larval instar, at day 3 after the molt, and reached a value of 13.5 nmol/min/mg. JH esterase was purified from hemolymph collected at this stage be a sequence of separation systems, including preparative nondenaturing PAGE, isoelectric focusing, and SDS-PAGE. The enzyme had a molecular weight of 120,000 and was composed of two subunits with molecular weights of 57,000, which were not linked by disulphide bridges. Isoelectric focusing revealed two forms of the enzyme with isoelectric points of 5.5 and 5.6. The Km and kcat of the purified enzyme were determined. The major form with pl 5.6 had a Km of 1.4 x 10(-6) M and a kcat of 0.9 s-1 and the minor form with pl 5.5 had a Km of 2.2 x 10(-6) M and a kcat of 1.9 s-1. The quaternary structure of L. decemlineata JH esterases, as differs from JH esterases in other species, which are monomers.

Expression of Escherichia coli branching enzyme in tubers of amylose-free transgenic potato leads to an increased branching degree of the amylopectin.

In order to increase the branching degree of potato tuber starch, the gene encoding branching enzyme (glgB) of Escherichia coli was expressed in the amylose-free potato mutant. The E. coli glgB was cloned in the binary vector pBIN19 under the transcriptional control of the potato Granule Bound Starch Synthase (GBSS) promoter and transitpeptide sequence. The E. coli glgB was cloned behind the two N-terminal amino acids of the GBSS mature protein, creating a chimeric protein. Transgenic plants were obtained which expressed the E. coli branching enzyme as was shown by the presence of mRNA and protein in the tubers. Correctly processed protein was found both in the soluble and starch granule bound protein fraction. Analysis of the starch showed an increase in the branching degree (DE) of up to 25% more branchpoints. The increase in the number of branchpoints was due to the presence of more short chains, with a degree of polymerization (DP) of 16 glucose-residues or less in the amylopectin. Changes in other characteristics of the starch, such as average chain length (CL) and lambda max, indicated a more branched structure for starch of transformed plants as well.