The complete sequence of the Cydia pomonella granulovirus genome.

The nucleotide sequence of the DNA genome of Cydia pomonella granulovirus (CpGV) was determined and analysed. The genome is composed of 123500 bp and has a G+C content of 45.2%. It contains 143 ORFs of 150 nucleotides or more that show minimal overlap. One-hundred-and-eighteen (82.5%) of these putative genes are homologous to genes previously identified in other baculoviruses. Among them, 73 are homologous to genes of Autographa californica nucleopolyhedrovirus (AcMNPV), whereas 108 and 98 are homologous to genes of Xestia c-nigrum GV (XcGV) and Plutella xylostella GV (PxGV), respectively. These homologues show on average 37.4% overall amino acid sequence identity to those from AcMNPV and 45% to those from XcGV and PxGV. The CpGV gene content was compared to that of other baculoviruses. Several genes reported to have major roles in baculovirus biology were not found in the CpGV genome, such as gp64, the major budded virus glycoprotein gene in some nucleopolyhedroviruses, and lef-7, involved in DNA replication. However, the CpGV genome encodes the large and small subunits of ribonucleotide reductase, three inhibitor of apoptosis (iap) homologues and two protein tyrosine phosphatases. The CpGV, PxGV and XcGV genomes present a noticeably high level of conservation of gene order and orientation. A striking feature of the CpGV genome is the absence of typical homologous repeat sequences. However, it contains one major repeat region and 13 copies of a single 73-77 bp imperfect palindrome.

Homologous expression of soluble methane monooxygenase genes in Methylosinus trichosporium OB3b.

An homologous expression system has been developed for soluble methane monooxygenase (sMMO) genes from Methylosinus trichosporium OB3b. sMMO-minus mutants were previously obtained after marker-exchange mutagenesis, by the insertion of a kanamycin-resistance cassette into the mmoX gene of the sMMO operon. Complementation of the sMMO-minus genotype was achieved by conjugation with broad-host-range plasmids containing the native promoter and sMMO operon from Ms. trichosporium OB3b (pVK100Sc and pHM2). In wild-type methanotrophs, copper ions present in the growth medium at concentrations greater than 0.25 microM inhibit transcription of sMMO genes. The stable maintenance of pVK100Sc resulted in transconjugant methanotrophs with a decreased sensitivity to copper, since expression of sMMO occurred at copper sulphate concentrations of 7.5 microM. sMMO activity was only detected in soluble extracts after the addition of purified sMMO reductase component, which is inhibited by copper ions in vitro. This phenomenon could have arisen due to the increased number of sMMO gene copies (derived from pVK100Sc) in the cell. Transconjugants obtained from conjugations with pHM2 expressed sMMO at copper concentrations of 0-2.5 microM only and sMMO activity was not restored by the addition of purified reductase component at copper concentrations higher than 2.5 microM. Southern hybridization showed that the plasmid had integrated into the chromosome, probably by a single homologous recombination event. This is the first report of homologous sMMO expression in a methanotroph with enzyme activities that are comparable to the activity reported in wild-type strains. This expression system will be useful for site-directed mutagenesis of active-site residues of sMMO from Ms. trichosporium OB3b.