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§725.421 Introduced genetic material.
For a new microorganism to qualify for either exemption under this subpart, introduced genetic material must meet all of the criteria listed in this section.
(a) Limited in size. The introduced genetic material must consist only of the following:
(1) The structural gene(s) of interest.
(2) The regulatory sequences permitting the expression of solely the gene(s) of interest.
(3) Associated nucleotide sequences needed to move genetic material, including linkers, homopolymers, adaptors, transposons, insertion sequences, and restriction enzyme sites.
(4) The nucleotide sequences needed for vector transfer.
(5) The nucleotide sequences needed for vector maintenance.
(b) Well-characterized. For introduced genetic material, well-characterized means that the following have been determined:
(1) The function of all of the products expressed from the structural gene(s).
(2) The function of sequences that participate in the regulation of expression of the structural gene(s).
(3) The presence or absence of associated nucleotide sequences and their associated functions, where associated nucleotide sequences are those sequences needed to move genetic material including linkers, homopolymers, adaptors, transposons, insertion sequences, and restriction enzyme sites.
(c) Poorly mobilizable. The ability of the introduced genetic material to be transferred and mobilized is inactivated, with a resulting frequency of transfer of less than 10-8 transfer events per recipient.
(d) Free of certain sequences. (1) The introduced genetic material must not contain a functional portion of any of the toxin-encoding sequences described in this paragraph (d).
(i) For the purposes of this section, a functional portion of a toxin-encoding sequence means any sequence which codes for a polypeptide that has one of the following effects:
(A) It directly or indirectly contributes to toxic effects in humans. Directly contributes to toxic effects in humans means those sequences encoding polypeptides that have direct toxicity to target cells. An example of a sequence which directly contributes to toxic effects in humans is one which encodes the portion of diphtheria toxin, listed in paragraph (d)(2) of this section, capable of interacting with elongation factor 2, leading to inhibition of protein synthesis in target respiratory, heart, kidney, and nerve tissues. Indirectly contributes to toxic effects in humans means a sequence whose encoded polypeptide is not directly toxic to target cells, yet still adversely affects humans. An example of a sequence which indirectly contributes to toxic effects is the sequence which encodes the portion of the botulinum toxin, listed in paragraph (d)(3) of this section, capable of blocking the release of acetylcholine from gangliosides. Botulinum toxin affects neuromuscular junctions by its blockage of acetylcholine release, leading to irreversible relaxation of muscles and respiratory arrest.
(B) It binds a toxin or toxin precursor to target human cells.
(C) It facilitates intracellular transport of a toxin in target human cells.
(ii) While these toxins are listed (with synonyms in parentheses) in paragraphs (d)(2) through (d)(7) of this section according to the source organism, it is use of the nucleotide sequences that encode the toxins that is being restricted and not the use of the source organisms. The source organisms are listed to provide specificity in identification of sequences whose use is restricted. Although similar or identical sequences may be isolated from organisms other than those listed below in paragraphs (d)(2) through (d)(7) of this section, these comparable toxin sequences, regardless of the organism from which they are derived, must not be included in the introduced genetic material.
(2) Sequences for protein synthesis inhibitor.
Sequence Source Toxin Name
Corynebacterium diphtheriae & C. Diphtheria toxin
ulcerans
Pseudomonas aeruginosa Exotoxin A
Shigella dysenteriae Shigella toxin (Shiga toxin,
Shigella dysenteriae type I
toxin, Vero cell toxin)
Abrus precatorius, seeds Abrin
Ricinus communis, seeds Ricin
(3) Sequences for neurotoxins.
Sequence Source Toxin Name
Clostridium botulinum Neurotoxins A, B, C1, D, E,
F, G (Botulinum toxins,
botulinal toxins)
Clostridium tetani Tetanus toxin
(tetanospasmin)
Proteus mirabilis Neurotoxin
Staphylococcus aureus Alpha toxin (alpha lysin)
Yersinia pestis Murine toxin
1 1 Snake toxins ............................
Bungarus caeruleus Caeruleotoxin
Bungarus multicinctus Beta-bungarotoxin
(phospholipase)
Crotalus spp. Crotoxin (phospholipase)
Dendroaspis viridis Neurotoxin
Naja naja varieties Neurotoxin
Notechia scutatus Notexin (phospholipase)
Oxyuranus scutellatus Taipoxin
Invertebrate toxins
Chironex fleckeri Neurotoxin
Androctnus australis Neurotoxin
Centruroides sculpturatus Neurotoxin
(4) Sequences for oxygen labile cytolysins.
Sequence Source Toxin Name
Bacillus alve Alveolysin
Bacillus cereus Cereolysin
Bacillus laterosporus Laterosporolysin
Bacillus thuringiensis Thuringiolysin
Clostridium bifermentans Lysin
Clostridium botulinum Lysin
Clostridium caproicum Lysin
Clostridium chauvoei Delta-toxin
Clostridium histolyticum Epsilon-toxin
Clostridium novyi Gamma-toxin
Clostridium oedematiens Delta-toxin
Clostridium perfringens Theta-toxin (Perfringolysin)
Clostridium septicum Delta-toxin
Clostridium sordellii Lysin
Clostridium tetani Tetanolysin
Listeria monocytogenes Listeriolysin (A B)
Streptococcus pneumoniae Pneumolysin
Streptococcus pyogene Streptolysin O (SLO)
(5) Sequences for toxins affecting membrane function.
Sequence Source Toxin Name
Bacillus anthracis Edema factor (Factors I II);
Lethal factor (Factors II
III)
Bacillus cereus Enterotoxin (diarrheagenic
toxin, mouse lethal factor)
Bordetella pertussis Adenylate cyclase (Heat-
labile factor); Pertussigen
(pertussis toxin, islet
activating factor,
histamine sensitizing
factor, lymphocytosis
promoting factor)
Clostridium botulinum C2 toxin
Clostridium difficile Enterotoxin (toxin A)
Clostridium perfringens Beta-toxin; Delta-toxin
Escherichia coli & other Heat-labile enterotoxins
Enterobacteriaceae spp. (LT); Heat-stable
enterotoxins (STa, ST1
subtypes ST1a ST1b; also
STb, STII)
Legionella pneumophila Cytolysin
Vibrio cholerae & Vibrio mimicus Cholera toxin (choleragen)
(6) Sequences that affect membrane integrity.
Sequence Source Toxin Name
Clostridium bifermentans & other Lecithinase
Clostridium spp
Clostridium perfringens Alpha-toxin (phospholipase
C, lecithinase);
Enterotoxin
Corynebacterium pyogenes & other Cytolysin (phospholipase C),
Corynebacterium spp. Ovis toxin
(sphingomyelinase D)
Staphylococcus aureus Beta-lysin (beta toxin)
(7) Sequences that are general cytotoxins.
Sequence Source Toxin Name
Adenia digitata Modeccin
Aeromonas hydrophila Aerolysin (beta-lysin,
cytotoxic lysin)
Clostridium difficile Cytotoxin (toxin B)
Clostridium perfringens Beta-toxin; Epsilon-toxin;
Kappa-toxin
Escherichia coli & other Cytotoxin (Shiga-like toxin,
Enterobacteriaceae spp. Vero cell toxin)
Pseudomonas aeruginosa Proteases
Staphylococcus aureus Gamma lysin (Gamma toxin);
Enterotoxins (SEA, SEB,
SEC, SED SEE); Pyrogenic
exotoxins A B; Toxic shock
syndrome toxins (TSST-1)
Staphylococcus aureus & Pseudomonas Leucocidin (leukocidin,
aeruginosa cytotoxin)
Streptococcus pyogenes Streptolysin S (SLS);
Erythrogenic toxins
(scarlet fever toxins,
pyrogenic exotoxins)
Yersinia enterocolitica Heat-stable enterotoxins
(ST)