We isolated and characterized CHO mutants deficient in peroxisome assembly using green fluorescent protein (GFP) and blue fluorescent protein (BFP) as the fluorescent probes to study the molecular mechanism of peroxisome biogenesis. We used stable transformants of CHO cells expressing GFP appending peroxisome targeting signal-1 (PTS1) and/or peroxisome targeting signal-2 (PTS2) as the parent strains for rapid isolation of the mutants. We have obtained six peroxisome-deficient mutants by visual screening of the mislocalizations of the peroxisomal GFPs. Mutual cell fusion experiments indicated that the six mutants isolated were divided into four complementation groups. Several of the mutants obtained possessed defective genes: the PEX2 gene was defective in SK24 and PT54; the PEX5 gene in SK32 and the PEX7 gene in PT13 and PT32. BE41, which belonged to the fourth complementation group, was not determined. When peroxisomal forms of BFP were transiently expressed in mutant cells, the peroxisomal BFPs appending both PTS1 and PTS2 appeared to bypass either the PTS1 or PTS2 pathway for localization in SK32. This observation suggested that other important machinery, in addition to the PTS1 or PTS2 pathway, could be involved in peroxisome biogenesis. Thus, our approach using peroxisomal fluorescent proteins could facilitate the isolation and analysis of peroxisome-deficient CHO mutants and benefit studies on the identification and role of the genes responsible for peroxisome biogenesis.
We have constructed a genome DNA map of the Antheraea pernyi nucleopolyhedrovirus (AnpeNPV) and used it to identify target genes for deletion in order to improve the newly developed baculovirus expression vector system. Initially, 50 independent PstI fragments of viral DNA were obtained by shotgun cloning, and both termini of each cloned fragment were sequenced. Then, the sequence data were used for homology search against both nucleotide and amino acid sequences of other NPVs in databases. This homology search allowed us to construct a nearly complete restriction map of a viral DNA with several assumed gaps. Four additional PstI fragments covering the gaps were obtained by PCR amplification, and a complete map of a circular viral DNA, which consisted of 54 PstI fragments, was constructed. The map indicated that the AnpeNPV genome is approximately 130.2 kbp in size and possesses high similarity to the Orgyia pseudotsugata multicapsid NPV (OpMNPV) genome in both sequence and arrangement of genes. Utilizing the genome-wide high similarity between AnpeNPV and OpMNPV, we identified two target genes on the map, namely, cathepsin and chitinase genes, whose products have been proved to be involved in the degradation of recombinant proteins and the liquefaction of virus-infected insect tissues. Comparative sequence analysis of the map also revealed the lack of certain OpMNPV open reading frame (ORF) homologs and the presence of ORFs, whose homologs do not exist in OpMNPV but in other group I NPVs, providing an insight into the position of AnpeNPV in the baculovirus phylogeny.
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