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Federal Register / Vol. 45, No. 227 / Friday, November 21, 1980 / Notices 
of the experiment is not required for 
most experiments described in Section 
III-O. Prior IBC review is required for all 
other experiments described in the 
subsections of Part III. 
Changes from the levels specified-in 
Part III for specific experiments (or the 
assignment of levels to experiments not 
explicitly considered here) may not be 
instituted without the express approval 
of the Director, NIH. (See Sections IV- 
E-l-b-(l)-(a), IV-E-l-b-(l)-(b), IV-E-1- 
b— (2)— (b), IV-E-l-b-(2)-(c), and IV-E-1- 
b-(3)-(b).) 
In the classification of containment 
criteria for different kinds of 
recombinant DNAs, the stated levels of 
physical and biological containment are 
minimal for the experiments designated. 
The use of higher levels of biological 
containment (HV3<HV2<HVl) is 
encouraged if they are available and 
equally appropriate for the purposes of 
the experiment. 
III-O. Classification of Experiments 
Using E. coli K-12 and Saccharomyces 
cerevisiae Host-Vector Systems. Most 
recombinant DNA experiments currently 
being done employ E. coli K-12 host- 
vector systems; others employ the S. 
cerevisiae host-vector systems. These 
are the systems for which we have the 
most experience and knowledge. 
Some experiments using E. coli K-12 
and S. cerevisiae host-vector systems 
and prohibited (see Section I-D). 
Some experiments using E. coli K-12 
and S. cerevisiae host-vector systems 
are exempt from the Guidelines (see 
Section I-E). 
Experiments using E. coli K-12 host- 
vector systems and DNA from Class 3 
organisms [1] or from cells known to be 
infected with these agents will be 
conducted at P3 containment or at a 
lower level as specified by NIH (See 
Section IV-E-l-b-2-(e)). 
Other experiments using E. coli K-12 
or laboratory strains of S. cerevisiae 
shall use PI physical containment and, 
except as specified in the last paragraph 
of this section, an HVl host-vector 
system [i.e., for experiments using E. 
■ coli K-12 (a) the E. coli host shall not 
contain conjugation-proficient plasmids 
or generalized transducing phages, and 
(b) lambda or lambdoid or Ff 
bacteriophages or non-conjugative 
plasmids [49] shall be used as vectors. 
For experiments in S. cerevisiae, 
laboratory strains shall be used]. For 
these experiments review by the IBC 
prior to the initiation of the experiment 
is not required. An exception, however, 
which does require prior review and 
approval by the IBC is any experiment 
in which there is a deliberate attempt to 
• have the E. coli K-12 efficiently express 
as a protein product the information 
carried in any gene derived from a 
eukaryotic organism or from any virus 
or viroid which infects a eukaryotic 
organism. 
Experiments involving the insertion 
into E. coli K-12 of DNA from 
prokaryotes that exchange genetic 
information with E. coli by known 
physiological processes will be 
exempted from these Guidelines if they 
appear on the "list of exchangers” set 
forth in Appendix A (see Section I-E-4). 
For those not on the Appendix A list 
but which exchange genetic information 
(35) with E. coli, experiments may be 
performed with any E. coli K-12 vector 
(e.g„ conjugafive plasmid). When a non- 
conjugative vector is used, the E. coli K- 
12 host may contain conjugation- 
proficient plasmids, either autonomous 
or integrated, or generalized transducing 
phages. 
III-Q-1. Experiments Involving Class 
3 Organisms. Experiments involving 
recombinant DNA from Class 3 
organisms [1) or from cells known to be 
infected with these agents may be 
conducted at P3 containment in E. coli 
K-12 EK1 hosts (see Section III-O). 
Containment levels for all other 
experiments with Class 3 organisms or 
with recombinant DNA which increases 
the virulence and host range of a plant 
pathogen beyond that which occurs by 
natural genetic exchange will be 
determined by NIH. (See Section (IV-E- 
l-b-2-(e)). 
• IU-A. Classification of Experiments 
Using Certain HVl and HV2 Host- 
Vector Systems. Certain HVl and HV2 
host-vector systems are assigned 
containment levels as specified in the 
subsections of this Section III— A. Those 
so classified as of publication of these 
revised Guidelines are listed in 
Appendix D. An updated list may be 
obtained from the Office of 
Recombinant DNA Activities, National 
Institutes of Health, Bethesda, Maryland 
20205. 
III-A-1. Shotgun Experiments. These 
experiments involve the production of 
recombinant DNAs between the vector 
and portions of the specified cellular 
source, preferably a partially purified 
fraction. Care should be taken either to 
preclude or eliminate contaminating 
microoganisms before isolating the 
DNA. 
III-A-l-a. Eukaryotic DNA 
Recombinants. 
Ill— A— 1— a— (1). Primates. P2 physical 
containment + an HV2 host- vector or 
P3 + HVl. 
III-A-l-a-(2). Other Mammals. P2 
physical containment + an HV2 host- 
vector or P3 + HVl. 
Ill— A— 1— a— ( 3 ) . Birds. P2 physical 
containment + an HV2 host-vector, or 
P3 + HVl. 
III-A-l-a-(4). Cold-Blooded 
Vertebrates. P2 physical containment 4- 
an HVl host-vector or Pi + HV2. If the 
eukaryote is known to produce a potent 
polypeptide toxin, [34) the containment 
shall be increased to P3 + HV2. 
III-A-l-a-(5). Other Cold-Blooded 
Animals and Lower Eukaryotes. This 
large class of eukaryotes is divided into 
two groups: 
III-A-l-a-(5)-a. Species that are 
known to produce a potent polypeptide 
toxin [34] that acts in vertebrates, or are 
known pathogens listed in Class 2, ( 1 ) or 
are known to carry such pathogens must 
use P3 physical containment -f an HV2 
host-vector. When the potent toxin is 
not a polypeptide and is likely not to be 
the product of closely linked eukaryote 
genes, containment may be reduced to 
P3 + HV1 or P2+HV2. Species that 
produce potent toxins that affect 
invertebrates or plants but not 
vertebrates require P2+HV2 or 
P3 + HV1. Any species that has a 
demonstrated capacity for carrying 
particular pathogenic microorganisms is 
included in this group, unless the 
organisms used a3 the source of DNA 
have been shown not to contain those 
agents, in which case they may be 
placed in the following group. [2A] 
III-A-l-a-(5)-(b). The remainder of 
the species in this class including plant 
pathogenic or symbiotic fungi that do 
not produce potent toxins: P2+HV1 or 
P1 + HV2. However, any insect in this 
group must be either (i) grown under 
laboratory conditions for at least 10 
generations prior to its use as a source 
of DNA, dr (ii) if caught in the wild, must 
be shown to be free of disease-causing 
microorganisms or must belong to a 
species that does not carry 
microorganisms causing disease in 
vertebrates or plants. [2A] If these 
conditions cannot be met, experiments 
must be done under P3+HV1 or 
P2+HV2 containment. 
III-A-l-a-{6). Plants. P2 physical 
containment + an HVl host-vector, or 
P1 + HV2. If the plant source makes a 
potent polypeptide toxin, [34) the 
containment must be raised to P3 
physical containment + HV2 host- 
vector. When the potent toxin is not a 
polypeptide and is likely not to be the 
product of closely linked plant genes, 
containment may be reduced to 
P3 + HV1 or P2 + HV2. [2 A) 
III-A-l-b. Prokaryotic DNA 
Recombinants. P2+HV1 or P1 + HV2 for 
experiments with phages, plasmids and 
DNA from nonpathogenic prokaryotes 
which do not produce polypeptide 
toxins. [34) P3 + HV2 for experiments 
[11] 
