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Molecular Genetic Characterization Data

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In July of 1998, regulatory officials of the Canadian Food Inspection Agency (CFIA), Health Canada, and the United States Department of Agriculture's Animal and Plant Health Inspection Service (USDA-APHIS) met to compare, and harmonize where possible, aspects of molecular genetic characterization that are part of their review processes for transgenic plants. Agreement on common requirements and acceptable analytical approaches for molecular genetic characterization will facilitate the submission of supporting data by developers seeking regulatory approval to incorporate such plants into agricultural production or commerce in both countries. This Appendix is one of the outcomes of this meeting. The Appendix summarizes and identifies similarities and differences in the critical elements of the molecular genetic characterization of transgenic plants considered during the review process by these participating agencies. Molecular genetic characterization is only part of the information considered during assessments of such plants conducted prior to commercialization.

The scope of this document is limited to consideration of the transformation process and vectors used during transformation; the genetic material that was potentially delivered to the recipient plant; the identification, inheritance, and expression of the genetic material in the transgenic plant, and the production of new proteins encoded by the introduced genetic material. This document does not address specific types of techniques nor quality assurance practices (e.g., good laboratory practices) that are used to generate molecular genetic characterization data.

The agencies found very substantial areas of agreement in the types of molecular genetic characterization data they require to be submitted and considered. In addition to the specific data sets reviewed, the participants of both countries reaffirmed that reviews are still conducted on a case-by-case basis which allows for reviewing additional or fewer data sets, depending upon the individual case and the regulatory authority of the individual agencies. The use of the word "may" in this document is intended to reflect some of this flexibility in determining when data sets will be considered as an appropriate part of the entire application package. Therefore, consultations between regulatory agencies and individual applicants are considered to be an important part of the overall application process in making such determinations.

The critical elements of the molecular genetic characterization of transgenic plants described below apply to the review process of the participating agencies in both Canada and the United States, except where noted. The contents of this document will be reviewed and amended as necessary by these agencies. The glossary which follows has been included to provide definition to certain terms within the context of this document.


carrier DNA
DNA used to expedite the preparation or the transformation of genetic material into a plant but which is itself not part of the construct.
coding region
A DNA sequence which can be translated to produce a protein. Synonymous with open reading frame.
An engineered DNA fragment (e.g. plasmid) which contains, but is not limited to, the DNA sequences to be integrated into a target plant's genome.
database citations
Publicly accessible sources of nucleotide or protein sequence information. Four commonly used databases and their website addresses are:

GenBank: An annotated collection of all publicly available DNA sequences maintained by the National Institute of Health (NIH).

DNA Data Bank of Japan: The officially certified DNA bank of Japan, which collects DNA sequences from researchers.

EMBL Nucleotide Sequence: A database of DNA and RNA sequences collected from the scientific literature, patent applications, and directly submitted from researchers and sequencing groups.

The SWISS-PROT Protein Sequence Data Bank: A database of protein sequences produced collaboratively by Amos Bairoch (University of Geneva) and the EBI.

That part of a construct (see above) which is integrated into the recipient plant's genome.
non-coding region
DNA sequences which lie outside of an open reading frame and which are not translated to become part of a protein. These might include scaffold attachment regions, promoters, leader sequences, enhancers, introns, terminators, and any other sequences that are used for gene expression either in the plant or other hosts. such as origins of replication, transposable elements, T-DNA borders, lox sequences, etc.
The ability of the transgenic trait to be expressed in the transformed plant line and plant lines derived therefrom in a consistent, reliable, and predictable manner.
The phenotypic characteristic(s) conferred to the recipient plant by the transgenic insert.
An autonomously replicating DNA molecule into which foreign DNA is inserted and then propagated in a host cell.

Molecular Genetic Characterization of Transgenic Plants

1. The Transformation System

2 Inheritance and stability of introduced traits which are functional in the plant

3 Characterization of the DNA Inserted in the Plant

4 Protein and RNA Characterization and Expression

Table 1: Example of a Table Describing the DNA Components of a Vector

Summary of DNA Components in PV-STBT02
(from Table III.1 from APHIS petition # 94-257-01p)
Genetic Element SizeTable Note 1
Function and Source
RB 0.36 A restriction fragment from the pTiT37 plasmid containing the 24 bp nopaline-type T-DNA right border used to initiate the T-DNA transfer from Agrobacterium tumefaciens to the plant genome (Depicker et al., 1982)
E35S 0.62 The cauliflower mosaic virus (CaMV) promoter (Odell et al., 1985) with the duplicated enhancer region (Kay et al., 1987).
cryIIIA 1.8 The gene which confers resistance to CPB. The gene encodes an amino acid sequence identical to the CPB control protein (referred to as the B.t.t. Band 3 protein) found in B.t.t. as described by Perlak et al. (1993).
E9 3' 0.63 A 3' nontranslated region of the pea ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) E9 gene (Coruzzi et al., 1984), which functions to terminate transcription and direct polyadenylation of the cryIIIA mRNA.
NOS 3' 0.26 A 3' nontranslated region of the nopaline synthase gene which functions to terminate transcription and direct polyadenylation of the nptII mRNA (Depicker et al., 1982; Bevan et al., 1983).
nptII 0.79 The gene isolated from Tn5 (Beck et al., 1982) which encodes for neomycin phosphotransferase type II. Expression of this gene in plant cells confers resistance to kanamycin and serves as a selectable marker for transformation (Fraley et al., 1983).
35S 0.32 The 35S promoter region of the cauliflower mosaic virus (CaMV) (Gardner et al., 1981; Sanders et al., 1987).
LB 0.45 A restriction fragment from the octopine Ti plasmid, pTi15955, containing the 24 bp T-DNA left border used to terminate the transfer of the T-DNA from Agrobacterium tumefaciens to the plant genome (Barker et al., 1983).
ori V 1.3 Origin of replication segment for ABI Agrobacterium derived from the broad- host range plasmid RK2 (Stalker et al., 1981).
ori-322/rop 1.8 A segment of pBR322 which provides the origin of replication for maintenance of the PV-STBT02 plasmid in E. coli, the replication of primer (rop) region and the bom site for the conjugational transfer into the Agrobacterium tumefaciens cells (Bolivar et al., 1977; Sutcliffe, 1978).
aad 0.93 A fragment isolated from transposon Tn7 containing a 0.79 kb gene which encodes for the enzyme streptomycin adenylyltransferase that allows for bacterial selection on spectinomycin or streptomycin (Fling et al., 1985).

Table Notes

Table Note 1

Sizes are approximations.

Return to table note referrer

Figure 1: Example of a detailed map of a plasmid vector

Example of a detailed map of a plasmid vector. Description follows.
Description for: Example of a detailed map of a plasmid vector

Restriction sites, and their locations in base pairs, utilized during Southern analyses are shown. The region which served as the T-DNA is marked and its delineating right and left borders are denoted by open arrows. The blackened regions denote the positions of homology for PCR probes used during Southern analysis Cleavage sites for HindIII, EcoRI, XhoI, and NotI restriction endonucleases are shown.

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