are designated EK1, EK2, and EK3 in 
ascending order. 
It has been necessary, throughout 
this section, to use words and phrases 
such as “purified” or “rigorously char- 
acterized." In the text such terms are 
marlced with footnote reference num- 
bers. These footnotes (part V) define 
more fully what these terms denote. 
In the following classification of con- 
tainment criteria for different kinds of 
recombinant DNA’s, the stated levels 
of physical and biological containment 
are minimal for the experiments desig- 
nated. The use of higher levels of bio- 
logical containment (EK3 > EK2 > 
EK1) is encouraged if they are availa- 
ble and equally appropriate for the 
purposes of the experiment. 
m-A-1. Shotgun Experiments. 
These experiments involve the produc- 
tion of recombinant DNA’s between 
the vector and portions of the speci- 
fied cellular source, preferably a par- 
tially purified fraction. Care should be 
taken either to preclude or eliminate 
contaminating microogranisms before 
isolating the DNA. 
ni-A-l-a Eukaryotic DNA Recom- 
binants. 
m-A-a-<l). Primates. P2 physical 
containment + and EK2 host-vector. 
Any lowering of containment below 
these levels CLe., for purified DNA or 
characterized clones) cannot be made 
solely by an institutional biosafety 
committee but requires NTH approval. 
H3-A-l-a-(2). Other Mammals. P2 
physical containment + an EK2 host- 
vector. 
IH-A-l-a-(3). Birds. P2 physical 
containment + an EK2 host-vector. 
m-A-l-*-(4). Cold-Blooded Verte- 
brates. P2 physical containment - an 
EK1 host-vector or PI + EK2. If the 
eukaryote is known to produce a 
potent polypeptide toxin, [ 34] the con- 
tainment shall be increased to P3 + 
EK2. 
m-A-l-a-<5). Other Cold-Blooded 
Animals and Lover Eukaryotes. This 
large class of eukaryotes is divided 
into two groups: 
IH-A-l-a-<5Ma). Species that are 
known to produce a potent polypep- 
tide toxinf 34) that acts in vertebrates, 
or are known pathogens listed in Class 
2,(1) or are known to carry such path- 
ogens must use P3 physical contain- 
ment - an EK2 host-vector. When the 
potent toxin is not a polypeptide and 
is likely not to be the product of close- 
ly linked eukaryote genes, contain- 
ment may be reduced to P3 + EK1 or 
P2 + EK2. Species that produce 
potent toxins that affect invertebrates 
of plants but not vertebrates require 
P2 + EK2 or P3 + EX1. Any species 
that has a demonstrated capacity for 
carrying particular pathogenic micro- 
organisms is included in this group, 
unless the organisms used as the 
source of DNA have been shown not to 
NOTICES 
contain those agents, in which case 
they may be placed in the following 
group. 
m-A-l-a-(5Mb). The remainder of 
the species in this class including 
plant pathogenic or symbiotic fungi 
that do not produce potent toxins: P2 
+ EK1 or PI + EK2. 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, or (11) if caught in 
the wild, must be shown to be free of 
dissease-ca using microorganisms or 
must belog to a species that does not 
carry microorganisms causing disease 
in vertebrates or plants. If these condi- 
tions cannot be met, experiments must 
be done under P3 + EK1 or P2 + EK2 
containment. 
m-A-l-a-(0). Plants. P2 physical 
containment + an EK1 host- vector or 
PI + EK2. If the plant source makes a 
potent polypeptide toxin, (14) the con- 
tainment must be raised to P3 physi- 
cal containment + an FK2 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 + 
EK1 or P2 + EK2. 
m-A-l-b. Prokaryotic DNA Recom- 
binants. 
III-A-l-b-(l). Prokaryotes That Ex- 
change Genetic Information's ) with 
E. Colt It is expected that many of 
the prokaryotes that exchange genetic 
information with E. coli by known 
physiological processes will be exempt- 
ed from these Guidelines by appearing 
on the ‘list of exchangers’ (see Section 
I-E-4). 
For those not o® the list, the con- 
tainment levels are PI physical con- 
tainment + an EK1 host-vector. In 
fact, experiments in this category may 
be performed with any E. coli K-12 
vector (e.g., coojugtive plasmids). How- 
ever, for prokaryotes that are classi- 
fied! I ) as Class 2 the containment 
levels are P2 + EK1. 
III-A-l-b-(2). Prokaryotes That Do 
Not Exchange Genetic Information 
With E. Colt P2 physical containment 
+ an EK1 ho6t-vector, or PI + EK2, 
except for DNA from Class 2 
agents/ 1) which require P3 + EK2. 
EU-A-2. Plasmids, Bacteriophages, 
and Other Viruses. Recombinants 
formed between a vector and some 
other plasmid or virus DNA have in 
common the potential for acting as 
double vectors because of the replica- 
tion functions in these DNA’s. The 
containment conditions given below 
apply only to propagation of the DNA 
recombinants in E. coli K-12 hosts. 
They do not apply to other hosts in 
which the recombinants may be able 
to replicate as a result of functions 
provided by the DNA inserted into the 
EK vectors. These are considered 
under other host-vector systems. 
' 33077 
m-A— 2— a. Viruses of Eukaryotes. 
(summary given in Table III). 
m-A-2-a-G). DNA Viruses. 
IH-A-2-a-GMa). Nontransforming 
viruses. 
m-A-2-a-(l)-<aMf). Adeno-Associ- 
ated Viruses, Minute Virus of Mice, 
Mouse Adenovirus ( strain PL), and 
Plant Viruses. PI physical contain- 
ment + and EK1 host-vector shall be 
used for DNA recombinants produced 
with (i) the whole viral genome, (il) 
subgenomlc DNA segments, or (ill) pu- 
rified cDNA copies of viral mRNA/ 3 7). 
m-A-2-a-(lMaM2). Hepatitis B. 
m-A-2-MlHs)-(2Wa). PI physical 
containment + an EK1 host-vector 
shall be used for purified subgenomlc 
DNA segments. 
m-A-2-a-< IMaMfMi). P2 physical 
containment + an EK 2 host-vector or 
P3 + EK1 shall be used for DNA for 
recombinants produced with the 
whole viral genome. 
m-A-2-a-(lMaM2Mc). P2 physical 
containment + an EK1, shall be used 
for DNA recombinants derived from 
purified cDNA copies of viral mRNA. 
III-A-2-a-( lWaMl). Other Non 
transforming Members of Presently 
Classified Viral Families.i36) 
IH-A-2-a-<lMaM3Ma). PI physical 
containment + an EK1 host-vector 
shall be used for (i) DNA recombin- 
ants produces with purified subgeno- 
mic DNA segments or (ii) purified 
cDNA copies of viral mRNA. (3 7) 
m-A-2-a-(lMaM3M6). PI physical 
cont ainm ent + an EK1 host and a 
vector certified for use in an EK2 
8 y stem shall be used for DNA recom- 
binants produced with the whole viral 
genome. 
HI-A-2-a-( 1 Mb). Transforming Vir- 
uses. 
Ed -A- 2-a-< 1Mb MI). Herpes Ssi- 
mirt Herpes A teles, and Epstein Barr 
Virus.139 ) 
m-A-2-a-UMbMIMo). PI physical 
cont ainm ent + an EK1 host-vectqr 
shall be used for DNA recombinants 
produced with purified nontrans- 
forming subgenamic DNA seg- 
ments/ 38) 
m-A-2-KlMb MIM6). P2 physical 
cont ainm ent + an EK1 host and a 
vector certified for use in an EK2 
system or P3 + EK1 shall be used for 
(i) DNA recombinants produced with 
purified subgenamic DNA segments 
containing an entire transforming 
gene or (ii) purified cDNA copies of 
viral mRNA/ 37) 
m-A-2-KlMbMlMc). P3 physical 
containment + an EK1 host-vector or 
P2 + EK2 shall be used for DNA re- 
combinants produced with the whole 
viral genome. 
IH-A-2-a-(lMBM3). Other Trans- 
forming Members of Presently Classi- 
fied Viral FamUies.(36) 
m-A-2-a-UMbM2Ma). PI physical 
containment + an EK1 host-vector 
FB>EkM ItEGtSTEt, VOL 43, NO. 146— PMBAY, JULY 26, 1476 
