60130 
38. >99% pure (i.e.. less than 1% of the 
DNA consists of intact viral genomes); oth- 
erwise as for whole genomes. 
39. The viruses have been classified by 
NCI as “moderate-risk oncogenic viruses.” 
See "Laboratory Safety Monograph— A Sup- 
plement to the NIH Guidelines for Recom- 
binant DNA Research” for recommenda- 
tions on handling the viruses themselves. 
40. EK1CV means the use of an EK1 host 
and a vector certified for use in an EK2 
system. 
41. The DNA preparation is defined as 
"purified" if the desired DNA represents at 
least 99% (w/w) of the total DNA in the 
preparation, provided that it was verified by 
more than one procedure. 
42. The lowering of the containment level 
when this degree of purification has been 
obtained is based on the fact that the total 
number of clones that must be examined to 
obtain the desired clone is markedly re- 
duced. Thus, the probability of cloning a 
harmful gene could, for example, be re- 
duced by more than 10-fold when a nonre- 
petitive gehe from mammals was being 
sought. Furthermore, the level of purity 
specified here makes it easier to establish 
that the desired DNA does not contain 
harmful genes. 
43. This is not permitted, of course, if it 
falls under any of the Prohibitions of Sec- 
tion I-D. Of particular concern here is pro- 
hibition I-D-5, i.e., "Deliberate transfer of a 
drug resistance trait to microorganisms that 
are not known to acquire 1 it naturally, if 
such acquisition could compromise the use 
of a drug to control disease agents in human 
or veterinary medicine or agriculture.” 
44. Because this work will be done almost 
exclusively in tissue culture cells, which 
have no capacity for propagation outside 
the laboratory, the primary focus for con- 
tainment is the vector. It should be pointed 
out that risk of laboratory-acquired infec- 
tion as a consequence of tissue culture ma- 
nipulation is very low. Given good microbio- 
logical practices, -the most likely mode of 
escape of recombinant DNAs from -a phys- 
ically contained laboratory is carriage by an 
infected human. Thus the vector with an in- 
serted DNA segment should have little or no 
ability to replicate or spread in humans. 
For use as a vector in a vertebrate host 
cell system, an animal viral DNA molecule 
should display the following properties: 
(i) It should not consist of the whole 
genome of any agent that is infectious for 
humans or that replicates to a significant 
extent in human cells in tissue culture. If 
the recombinant molecule is used to trans- 
form nonpermissive cells (i.e., cells which do 
not produce infectious virus particles), this 
is not a requirement. 
(ii) It should be derived from a virus 
whose epidemiological behavior and host 
range are well understood. 
(iii) In permissive cells, it should be defec- 
tive when carrying an inserted DNA seg- 
ment (i.e., propagation of the recombinant 
DNA as a virus must be dependent upon the 
presence of a complementing helper 
genome). In almost all cases this condition 
would be achieved automatically by the ma- 
nipulations used to construct and propagate 
the recombinants. In addition, the amount 
of DNA encapsulated in the particles of 
most animal viruses is defined within fairly 
close limits. The insertion of sizable foreign 
DNA sequences, therefore, generally de- 
mands a compensatory deletion of viral se- 
quences. It may be possible to introduce 
FEDERAL 
NOTICES 
very short insertions (50-100 base pairs) 
without rendering the viral vector defective. 
In such a situation, the requirement that 
the viral vector be defective is not neces- 
sary, except in those cases in which the in- 
serted DNA encodes a biologically active po- 
lypeptide. 
It is desired but not required that the 
functional anatomy of the vector be 
known— that is, there should be a clear idea 
of the location within the molecule of: 
(i) The sites at which DNA synthesis origi- 
nates and terminates, 
(ii) The sites that are cleaved by restric- 
tion endonucleases, and 
(iii) The template regions for the major 
gene product. 
If possible the helper virus genome 
should: (i) Be integrated into the genome of 
a stable line of host cells (a situation that 
would effectively limit the growth of the 
vector recombinant to such cell lines) or 
(ii) Consist of a defective genome, or an 
appropriate conditional lethal mutant virus, 
making vector and helper dependent upon 
each other for propagation. 
However, neither of these stipulations is a 
requirement. 
45. Review by NIH on a case-by-case basis 
means that NIH must review and set appro- 
priate containment conditions before the 
work may be undertaken. NIH actions m 
such case-by-case reviews will be published 
in the Recombinant DNA Technical Bulle- 
tin. 
46. Provided the inserted DNA sequences 
are not derived from eukaryotic viruses. In 
the latter case, such experiments will be 
evaluated on a case^by-case basis. 
47. .*99% pure; otherwise as for shotgun 
experiments. 
Appendix A 
Section I-E-4 states 'that exempt from 
these Guidelines are "certain specified re- 
combinant DNA molecules that consist en- 
tirely of DNA segments from different spe- 
cies that exchange DNA by -known physio- 
logical -processes, though one or more of the 
segments may be a synthetic equivalent. A 
list of such exchangers will be prepared and 
periodically revised by the Director, NIH, 
with advice of the Recombinant DNA Advi- 
sory Committee, after appropriate notice 
and opportunity for public comment (see 
Section IV-E-l-b-G)-(d).) Certain classes 
are exempt as of publication of these Re- 
vised Guidelines. The list is in Appendix A.” 
Under exemption I-E-4 of these revised 
Guidelines are recombinant DNA molecules 
that are (1) composed entirely of DNA seg- 
ments from one or more of the organisms of 
the following classes, and (2) to be propagat- 
ed in any of the organisms listed below. 
(Classification of Bergey’s Manual of Deter- 
minative Bacteriology, eighth edition. R. E. 
Buchanan and N. E. Gibbons, editors. Wil- 
liams and Wilkins Company: Baltimore, 
1974.) 
1. Genus Escherichia 
2. Genus Shigella 
3. Genus Salmonella (including Arizona) 
4. Genus Enterobacter 
5. Genus Citrobacter (including Levinea ) 
6. Genus Klebsiella 
7. Erwinia amylovora 
8. Pseudomonas aeruginosa 
9. Serratia marcescens 
REGISTER, VOL 43, NO. 247— FRIDAY, DECEMBER 
[52] 
Appendix B 
CLASSIFICATION OF MICROORGANISMS ON 
THE BASIS OF HAZARD 
I. Classification of Etiologic Agents on the 
Basis of Hazard ( 1 ) 
A. Class 1 Agents 
All bacterial, parasitic, fungal, viral, rick- 
ettsial. and chlamydial agents not included 
in higher classes. 
B. Class 2 Agents 
1. Bacterial Agents 
Actinobacillus— all species except, A. mallei, 
which is in Class 3 
Arizona hinshawii— all serotypes 
Bacillus anthracis 
Bordetella—al 1 species 
Borrelia recurrentis, B. vincenti 
Clostridium botulinum, Cl. chauvoei, Cl. 
haemolyticum, CL histolyticum, Cl. novyi. 
Cl. septicum, CL tetani 
Corynebacterium diptheriae, C. equi, C. hae- 
molyticum, C. pseudotuberculosis, C. pyo- 
genes, C. renale 
Diplococcus ( Streptococcus ) pneumoniae 
Erysipelothrix insidiosa 
Escherichia coli— all enteropathogenic sero- 
types 
Haemophilus ducreyi, H. influenzae 
Herellae vaginicola 
Klebsiella— all species and all serotypes 
Leptospira interrogans— all serotypes 
Listeria— all species 
Mima polymorpha 
Moraxella — all species 
Mycobacteria— all species except those 
listed in Class 3 
Mycoplasma— all species except Myco- 
plasma mycoides and Mycoplasma agalac- 
tiae, which are in Class 5 
Neisseria gonorrhoeae, N. meningitidis 
Pasteurella—all species except those listed 
in Class 3 
Salmonella — all -species and all serotypes 
Shigella— all species and all serotypes 
Sphaerophorus necrophorus 
Staphylococcus aureus 
Streptobadllus moniliformis 
Streptococcus pyogenes 
Treponema carateum, T. pallidum, and T. 
pertenue 
Vibrio fetus, V. comma, including biotype El 
Tor, and V. parahemolyticus 
2. Fungal Agents 
1 Actinomycetes (including Nocardia species 
and Actinomyces species and Arachnia 
propionica ) 
Blastomyces dermatitidis 
Cryptococcus neoformans 
Paracoccidioides brasiliensis 
3. Parasitic Agents 
Endamoeba histolytica 
Leishmania sp. 
Waegleria gruberi 
Toxoplasma gondii 
Toxocara canis 
Trichinella spiralis 
Trypanosoma cruzi 
4. Viral, RickettsiaL and Chlamydial Agents 
Adenoviruses— human— all types 
Cache Valley virus 
' Since the publication of the classification 
in 197-4 Cl], the Actinomycetes have been re- 
classified as bacterial rather than fungal 
agents. 
22, 1978 
