40 C.F.R. § 725.421

Current through October 31, 2024
Section 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 SourceToxin Name
Corynebacterium diphtheriae & C. ulceransDiphtheria toxin
Pseudomonas aeruginosaExotoxin A
Shigella dysenteriaeShigella toxin (Shiga toxin, Shigella dysenteriae type I toxin, Vero cell toxin)
Abrus precatorius, seedsAbrin
Ricinus communis, seedsRicin

(3)Sequences for neurotoxins.

Sequence SourceToxin Name
Clostridium botulinumNeurotoxins A, B, C1, D, E, F, G (Botulinum toxins, botulinal toxins)
Clostridium tetaniTetanus toxin (tetanospasmin)
Proteus mirabilisNeurotoxin
Staphylococcus aureusAlpha toxin (alpha lysin)
Yersinia pestisMurine toxin
Snake toxins
Bungarus caeruleusCaeruleotoxin
Bungarus multicinctusBeta-bungarotoxin (phospholipase)
Crotalus spp.Crotoxin (phospholipase)
Dendroaspis viridisNeurotoxin
Naja naja varietiesNeurotoxin
Notechia scutatusNotexin (phospholipase)
Oxyuranus scutellatusTaipoxin
Invertebrate toxins
Chironex fleckeriNeurotoxin
Androctnus australisNeurotoxin
Centruroides sculpturatusNeurotoxin

(4)Sequences for oxygen labile cytolysins.

Sequence SourceToxin Name
Bacillus alveAlveolysin
Bacillus cereusCereolysin
Bacillus laterosporusLaterosporolysin
Bacillus thuringiensisThuringiolysin
Clostridium bifermentansLysin
Clostridium botulinumLysin
Clostridium caproicumLysin
Clostridium chauvoeiDelta-toxin
Clostridium histolyticumEpsilon-toxin
Clostridium novyiGamma-toxin
Clostridium oedematiensDelta-toxin
Clostridium perfringensTheta-toxin (Perfringolysin)
Clostridium septicumDelta-toxin
Clostridium sordelliiLysin
Clostridium tetaniTetanolysin
Listeria monocytogenesListeriolysin (A B)
Streptococcus pneumoniaePneumolysin
Streptococcus pyogeneStreptolysin O (SLO)

(5)Sequences for toxins affecting membrane function.

Sequence SourceToxin Name
Bacillus anthracisEdema factor (Factors I II); Lethal factor (Factors II III)
Bacillus cereusEnterotoxin (diarrheagenic toxin, mouse lethal factor)
Bordetella pertussisAdenylate cyclase (Heat-labile factor); Pertussigen (pertussis toxin, islet activating factor, histamine sensitizing factor, lymphocytosis promoting factor)
Clostridium botulinumC2 toxin
Clostridium difficileEnterotoxin (toxin A)
Clostridium perfringensBeta-toxin; Delta-toxin
Escherichia coli & other Enterobacteriaceae spp.Heat-labile enterotoxins (LT); Heat-stable enterotoxins (STa, ST1 subtypes ST1a ST1b; also STb, STII)
Legionella pneumophilaCytolysin
Vibrio cholerae & Vibrio mimicusCholera toxin (choleragen)

(6) Sequences that affect membrane integrity.

Sequence SourceToxin Name
Clostridium bifermentans & other Clostridium sppLecithinase
Clostridium perfringensAlpha-toxin (phospholipase C, lecithinase); Enterotoxin
Corynebacterium pyogenes & other Corynebacterium spp.Cytolysin (phospholipase C), Ovis toxin (sphingomyelinase D)
Staphylococcus aureusBeta-lysin (beta toxin)

(7)Sequences that are general cytotoxins.

Sequence SourceToxin Name
Adenia digitataModeccin
Aeromonas hydrophilaAerolysin (beta-lysin, cytotoxic lysin)
Clostridium difficileCytotoxin (toxin B)
Clostridium perfringensBeta-toxin; Epsilon-toxin; Kappa-toxin
Escherichia coli & other Enterobacteriaceae spp.Cytotoxin (Shiga-like toxin, Vero cell toxin)
Pseudomonas aeruginosaProteases
Staphylococcus aureusGamma lysin (Gamma toxin); Enterotoxins (SEA, SEB, SEC, SED SEE); Pyrogenic exotoxins A B; Toxic shock syndrome toxins (TSST-1)
Staphylococcus aureus & Pseudomonas aeruginosaLeucocidin (leukocidin, cytotoxin)
Streptococcus pyogenesStreptolysin S (SLS); Erythrogenic toxins (scarlet fever toxins, pyrogenic exotoxins)
Yersinia enterocoliticaHeat-stable enterotoxins (ST)

40 C.F.R. §725.421