Points to consider in seeking biosafety approval for research, testing, and environmental release of experimental genetically modified biocontrol products during research and development.

Novel genetically modified biological control products (referred to as "GM biocontrol products") are being considered to address a range of complex problems in public health, conservation, and agriculture, including preventing the transmission of vector-borne parasitic and viral diseases as well as the spread of invasive plant and animal species. These interventions involve release of genetically modified organisms (GMOs) into the environment, sometimes with intentional dissemination of the modification within the local population of the targeted species, which presents new challenges and opportunities for regulatory review and decision-making. Practices developed for GMOs, primarily applied to date for GM crops may need to be adapted to accommodate different types of organisms, such as insects, and different technologies, such as gene drive. Developers of new GM biocontrol products would benefit from an early understanding of safety data and information that are likely to be required within the regulatory dossier for regulatory evaluation and decision making. Here a generalizable tool drawing from existing GM crop dossier requirements, forms, and relevant experience is proposed to assist researchers and developers organize and plan their research and trialing. This tool requires considering specifics of each investigational product, their intended use, and country specific requirements at various phases of potential product development, from laboratory research through contained field testing and experimental release into the environment. This may also be helpful to risk assessors and regulators in supporting their systematic and rigorous evaluation of new biocontrol products.

Watermelon mosaic virus II and zucchini yellow mosaic virus: cloning of 3'-terminal regions, nucleotide sequences, and phylogenetic comparisons.

The 3'-terminal genomic regions of an isolate of watermelon mosaic virus II (WMVII) and a Florida isolate of zucchini yellow mosaic virus (ZYMV-F) have been cloned. The nucleotide sequence of the WMVII cDNA clone shows the presence of the large nuclear inclusion protein gene, the coat protein gene and 3' untranslated region. The nucleotide sequence of a ZYMV-F cDNA clone shows the presence of the coat protein gene and 3' untranslated region. Comparisons of the nucleotide and deduced amino acid sequences of these clones with those from other potyviruses show that WMVII and the soybean mosaic virus N strain are closely related, thus supporting their classification as different strains of the same virus. Our comparisons also indicate that ZYMV-F is a distinct potyvirus type and that its closest relative is WMVII. Phylogenetic analysis using the most-parsimonious branching arrangement derived from the alignment of coat protein gene sequences suggests the existence of two major potyvirus groupings.

The nucleotide sequences of the 3'-terminal regions of papaya ringspot virus strains W and P.

The sequences of cDNA clones encoding most of the NIb protein, the coat protein and the 3' untranslated region of papaya ringspot virus (PRV) strains W and P have been determined. The open reading frame of P strain PRV was confirmed by amino acid analysis. Nucleotide sequence comparisons of these strains show that they share a 98.2% identity in their NIb gene regions and a 97.7% identity in their coat protein genes. The sequences of these two strains are distinct from other potyvirus types, confirming their classification as two strains of the same virus. The NIb amino acid sequence possesses conserved amino acids characteristic of RNA-dependent RNA polymerases. Comparison of the coat protein amino acid sequence with those of other potyviruses shows perfectly conserved amino acids which may have functional significance.

Nucleotide sequences of the coat protein genes and flanking regions of cucumber mosaic virus strains C and WL RNA 3.

Several strains of cucumber mosaic virus (CMV) have been classified, and nucleic acid hybridization data indicate that these strains differ widely in nucleotide sequence. We have constructed cDNA clones of the coat protein coding regions of CMV strains C and WL, and have compared the nucleotide sequences of the RNA 3 intergenic region, coat protein gene, and 3' untranslated region with published CMV sequences from the same regions of the Q, D and Y strains. These comparisons show that the C and WL strains belong to different CMV subgroups, and that the subgroups are more closely related in sequence than suggested by previous nucleic acid hybridization studies.

Changes in methylation of tissue cultured soybean cells detected by digestion with the restriction enzymes HpaII and MspI.

Each of the tandemly arranged 5S RNA genes of soybean contain two CCGG sites which, if unmethylated, can be digested by both MspI and HpaII. Methylation of the internal cytosine (CmeCGG) prevents digestion by HpaII but allows digestions by MspI.Suspension cultures were prepared from soybean plants and the DNA from these cultures was examined for the susceptibility of 5S RNA genes to digestion by MspI and HpaII. 5S genes from DNA extracted from intact plants can be partially digested with MspI but not at all by HpaII. In contrast, shortly after cells were cultured the 5S RNA could be hydrolyzed by both HpaII and MspI. After prolonged cell culture, the 5S genes from some cell lines were found to have become partially or even completely resistant to HpaII digestion. The results suggest that lack of methylation can occur when cells are cultured and that such methylation may play a role in the heritable changes observed in cell culture.