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Appendix I:
Molecular Genetic Characterization Data
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INTRODUCTION
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.
GLOSSARY
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. |
construct |
An engineered DNA fragment (eg. 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).
http://www.ncbi.nlm.nih.gov/Genbank/GenbankOverview.html
DNA Data Bank of Japan: The officially certified DNA bank
of Japan, which collects DNA sequences from researchers. http://www.ddbj.nig.ac.jp/fromddbj-e.html
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. http://www.ebi.ac.uk/embl/
The SWISS-PROT Protein Sequence Data Bank: A database of
protein sequences produced collaboratively by Amos Bairoch (University of
Geneva) and the EBI. http://www.ebi.ac.uk/swissprot/
|
insert |
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. |
stability |
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. |
trait(s) |
The phenotypic characteristic(s) conferred to the recipient
plant by the transgenic insert. |
vector |
An automonously 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 |
1.1 |
Description of the transformation method |
1.1.1 |
Describe and provide references for the transformation method,
e.g. Agrobacterium- mediated transformation or direct transformation
by methods such as particle bombardment, electroporation, PEG transformation of
protoplasts, etc. |
1.1.2 |
For direct transformation methods, describe the nature and
source of any carrier DNA used. |
1.1.3 |
For Agrobacterium-mediated transformation, provide the
strain designation of the Agrobacterium used during the transformation
process, and indicate how the Ti plasmid based vector was disarmed, and whether
Agrobacterium was cleared from the transformed tissue. |
1.1.4 |
For transformation systems other than Agrobacterium,
provide the following information: |
1.1.4.1 |
Does the system utilize a pathogenic organism or nucleic acid
sequences from a pathogen? |
1.1.4.2 |
How were any pathogenesis-related sequences removed prior to
transformation? |
1.1.4.3 |
Did the transformation process involve the use of helper
plasmids or a mixture of plasmids? If so, describe these in detail. |
1.2 |
Description of the genetic material potentially delivered to the
recipient plant material (the modification/constructs). |
1.2.1 |
Provide a summary of all genetic components which comprise the
vector including coding regions, and non-coding sequences of known function
(see Table 1). For each genetic component provide a citation where these
functional sequences were described, isolated, and characterized (publicly
available database citations are acceptable) and indicate: |
1.2.1.1 |
The portion and size of the sequence inserted. |
1.2.1.2 |
The location, order, and orientation in the vector. |
1.2.1.3 |
The function in the plant. |
1.2.1.4 |
The source (scientific and common, or trade name, of the donor
organism). |
1.2.1.5 |
If the genetic component is responsible for disease or injury to
plants or other organisms, and is a known toxicant, allergen, pathogenicity
factor, or irritant. |
1.2.1.6 |
If the donor organism is responsible for any disease or injury
to plants or other organisms, produces toxicants, allergens or irritants or is
related to organisms that do. |
1.2.1.7 |
If there is a history of safe use of the source organism or
components thereof. |
1.2.2 |
If there has been a significant modification that affects the
amino acid sequence of genes designed to be expressed in the plant, provide the
citation. If the modified amino acid sequence has not been published, provide
the complete sequence highlighting the modifications. Modifications that affect
only a few amino acids can simply be stated without providing the complete
sequence. Indicate whether the modifications are known or expected to result in
changes in post-translational modifications or sites critical to the structure
or function of the gene product. |
1.2.3 |
Provide a detailed map of the vector (see Figure 1) with the
location of sequences described above that is sufficient to be used in the
analysis of data supporting the characterization of the DNA, including as
appropriate the location of restriction sites and/or primers used for PCR and
regions used as probes.
|
2 |
Inheritance and stability of introduced traits which are
functional in the plant |
2.1 |
For plants which are either male or female fertile or both,
provide data that demonstrates the pattern and stability of inheritance and
expression of the new transgene traits. If the new trait can not be directly
measured by an assay, it may be necessary to examine the inheritance of the DNA
insert directly, and expression of the RNA. |
2.2 |
For plants which are either infertile or for which it is
difficult to produce seed (such as vegetatively propagated male-sterile
potatoes), provide data to demonstrate that the transgene trait is stably
maintained and expressed during vegetative propagation over a number of cycles
that is appropriate to the crop.
|
3 |
Characterization of the DNA Inserted in the
Plant |
3.1 |
For all coding regions, provide data that
demonstrate if complete or partial copies are inserted into the plant's
genome. Coding regions may include truncated sense constructs, sequences
engineered to be nontranslatable, antisense constructs, and constructs
containing ribozymes, regardless of whether or not the coding region is
designed or expected to be expressed in the transgenic plant. For Canadian
submissions, information may be required indicating the number of copies which
have been inserted, including integration of partial copies; and for
allopolyploid plants, information indicating into which parental genome
insertion has occurred. |
3.2 |
For noncoding regions associated with the expression of coding
regions: |
3.2.1 |
Data should demonstrate whether or not plant promoters are
inserted intact with the coding regions whose expression they are designed to
regulate. |
3.2.2 |
DNA analysis may be necessary for introns, leader sequences,
terminators, and enhancers of plant-expressible cassettes. |
3.2.3 |
DNA analysis may be necessary for promoters and other regulatory
regions associated with bacteria-expressible cassettes. |
3.3 |
For noncoding regions which have no known plant
function and are not associated with expression of coding regions: |
3.3.1 |
DNA analysis may be required for some sequences of known
function (e.g., ori V
and ori-322, bom, T-DNA
borders of Agrobacterium, and bacterial transposable elements). |
3.3.2 |
DNA analysis is not required for any remaining sequences of the
plasmid backbone.
|
4 |
Protein and RNA Characterization and Expression
|
4.1 |
For all complete coding regions inserted,
provide data that demonstrates whether the protein is or is not produced as
expected in the appropriate tissues consistent with the associated regulatory
sequences driving its expression (e.g., if the gene is inducible, determine if
the gene is expressed in the appropriate tissues under induction conditions).
For virus resistant plants where the transgenes are derived from a viral
genome, in addition to transgene protein analysis, determine transgene RNA
levels in tissues consistent with the associated regulatory regions driving
expression of the transgene. The following exceptions also apply: |
4.1.1 |
If the protein concentration is below the limits of detection,
mRNA data may be substituted. |
4.1.2 |
Protein analysis for products of genes used only as selectable
markers may be waived under certain circumstances, e.g. when there is at least
one complete copy of a selectable marker gene present and the effective
expression of the selectable marker gene is verified by the process used to
select the transformed tissue. |
4.1.3 |
For plants modified to express nontranslatable mRNA, truncated
sense constructs, antisense constructs, or constructs containing ribozymes,
since the function of these genetic constructs is to specifically alter the
accumulation of a specific mRNA or protein present in the transgenic plant,
provide data on the level of the target protein only (e.g.
native tomato fruit polygalacturonase would be the target protein of antisense
polygalacturonase to achieve altered fruit ripening). If the target protein
levels are below levels of detection, determine target mRNA levels. |
4.2 |
When a fragment of a coding region designed to be expressed in a
plant is detected, determine whether a fusion protein could be produced and in
which tissues it may be located. |
4.3 |
Protein or RNA characterization may not be required for
fragments of genetic constructs not expected to be functional in the
plant (e.g., fragments of selectable marker genes driven by bacterial
promoters.) |
Summary of DNA Components in PV-STBT02 (from Table III.1 from APHIS
petition # 94-257-01p)
Genetic Element |
Size1
Kb |
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). |
1. Sizes are approximations.
Figure 1: Example of a detailed map of a plasmid vector
[D]
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