33088 
NOTICES 
18 Runkle. R. S.. and G. B Phillips 
(1969). Microbial Containment Control Fa- 
cilities. Van Non trend Reinhold. Sri/ York. 
19. Chattgny. M. A., and D I. Cling»r 
(1969). Contatmination Control in Aerobio- 
logy In R. L. Dimmick and A B Akers' 
(eds.) An Introduction to Experimental Aer- 
obiology. John Wiley Ac Sons. Near York pp. 
194-263 
20. Biological safety cabinets referred to 
In this section are classified as class I. clou 
II. or class III cabinets. A class l is a venti- 
lated cabinet for personnel protection 
having an Inward flow of air away from the 
operator. The exhaust air from this cabinet 
Is filtered through a high-efficiency particu- 
late air (HEPA) filter. This cabinet is used 
in three operational modes (1) with a full- 
width open front. (2) with an Installed front 
closure panel (having four 8-tnch diameter 
openings) without gloves, and (I) with an In- 
stalled front closure panel equipped with 
arm-length rubber glovea The face velocity 
of the inward flow of air through the full- 
width open front Is 75 feet per minute or 
greater. A class II cabinet Is a ventilated 
cabinet tor personnel and product protec- 
tion having an open front with Inward air 
flow for personnel protection, and HEPA fil- 
tered mass recirculated air flow for product 
protection. The cabinet exhaust air Is fil- 
tered through s HEPA filter. The face ve- 
locity of the inward flow of air through the 
full-width open front is 75 feet per minute 
or greater Design and performance speclfl- 
eatiora for class II cabinet* have been 
adopted by the National Sanitation Founda- 
tion. Ann Arbor. Mich. A class III cabinet is 
a closed-front ventilated cabinet of gas-tight 
construction which provides the highest 
level of personnel protection of all bloha- 
zard safely cabinets The Interior of the 
cabinet is protee'ed from contaminants ex- 
terior to the cabinet. The cabinet is fitted 
with arm-length rubber gloves and Is operat- 
ed under a nega>lve pressure of at least 0.5 
inch water gauge. All supply air is filtered 
through HEPA filters Exhaust air is fil- 
tered through two HEPA filters or one 
HEPA filter and tncinerator before being 
discharged to the outside environment. 
21. Hrrahfield. V . H W Boyer. C. Yan- 
ofsky. M. A Lovett, and D. R. He Unski 
(1974). Plasmid Col FI as a Molecular Vehi- 
cle for Coning and Amplification of DMA. 
Proc. Nat. Acad Set USA 11. 3455 3459. 
22. Wensink. P C.. D. J. Finnegan. J. E 
Done Ison and D. S. Hogness (1974). A 
System for Mapping DNA Sequence* In the 
Chromosomes of Drosophila Melanogaster. 
Cell l 315-235 
23 Tanaka T.. and B Weisblum (1975). 
Construction of a Colicin FI R Factor Com- 
posite Plasmid In Vitro Means for Amplica- 
tion of Deosymbonucletc Acid. J. BactertoL 
121. 354 362. 
24 Amstrong. K. A.. V. Hershfield. and D. 
R. Helinski 0977). Gene Cloning and Con- 
tainment Properties of Plasmid Col £1 and 
Its Derivatives. Science I9t. 172-174. 
25 Bolivar. P.. R U Rodriguez. M. C. Bet- 
lach. and H. W. Boyer (1977). Construction 
and Charade -nation of Sac Cloning VeAt- 
cles: I. AmplciUin-Reslstant Derivative of 
pMBt. Gene, in press. 
26. Cohen. S. W A. C. W. Chang. H. 
Boyer, and R. Helling (1973). Construction 
of Biologically Functional Barterial Plas- 
mids tn Vlfro. Proc. NaU. Acad. Set USA 70. 
3240-3244 
27. Bolivar. F., R L. Rodriguez. R. J. 
Greene. M C. Ballach. H. L. Reyneker. H. 
W Boyer. J. H. Crosa. and S. Fallcow. Con- 
struction and Characterization of Neve 
Cloning Vehicles: II. A Multi-Purpose Clon- 
ing System. Gene, in press. 
28. Thomas. M., J R. Cameron, and R. W 
Davis 0974). Viable Molecular Hybrids of 
Bacteriophage Lambda and Eukaryotic 
DNA. Proc. Nat. Acad. Set USA 71. 4579- 
43583 
29 Murray. N. E.. and K. Murray 0974). 
Manipulation of Restriction Targets in 
Phage Lambda to Form Receptor Chromo- 
somes for DNA Fragments Nature 251. 476- 
481. 
30. Ram bach. A., and P. Tiollais (1974). 
Bacteriophage Lambda Having EcoRI En- 
donuclease Sites Only in the Non-Essential 
Region of the Genome. Proc. Nat. Acad. Sci. 
USA 71 3927-3930 
31 Blaltner. P R B O Williams. A. E 
Bleche. K Dcnnlston-Thompson. H. E 
Faber L. A. Furlong D. J. Gunwaid. D. O. 
Kle/er. D. D Moore. J W. SUumm. E. L. 
Sheldon. and O Smithies 0977). Charon 
Phages Safer Derivatives of Bacteriophage 
Lambda for DNA Cloning. Science 196. 163- 
169 
32. Donoghue. D. J.. and P A Sharp 
(1977). An Improved Lambda Vector Con- 
struction of Model Recombinants Coding for 
Kmamyctn Resistance. Gene I. 209-227 
33 Leder. P.. D. Tlemeier and L. Enquist 
0977). EK2 Derivatives of Bacteriophage 
Lambda Useful in the Cloning of DNA from 
Higher Organisms The gt WES System. Sci- 
ence 194.175-177. 
34. We are specifically concerned with 
potent toxins which could be produced by 
acquiring a tingle gene or cluster of genes. 
35. Defined as observable under optimal 
laboratory conditions by transformation, 
transduction, phmge infection, and or conju- 
gation with transfer of phage, plasmid, and/ 
or chromosomal genetic information. Note 
that this definition of exchange may be less 
stringent than that applied to exempt or- 
ganisms under mcuoe I E 4 
36. As classified in the Second Report of 
the International Committee on Taxonomy 
of Viruses: Clssiwflcwnon and Nomenclature 
of Viruses. Prank Psnner. Ed. Intervirology 
7(19-115) 1976 (As noted m the Prohibition 
Section, the use of viruses classified! 1 ] as 
class 3. 4. or 5. other than VSV. is prohibit- 
ed.) 
37. The cDNA copy of the viral mRNA 
must be >99 percent pure; otherwise as for 
shotgun experiments with eukaryotic cellu- 
lar DNA. 
38. >99 percent pure (l.e.. less than 1 per- 
cent of the DNA consists of Intact viral gen- 
ames); otherwise as for whole genaraes. 
39. The viruses have been classified by 
NCI as moderate risk oncogenic viruses " 
8ee Laboratory Safety Monograph— A Sup- 
plement to the NIH Guidelines for Recom- 
binant DNA Research" for recomendattons 
on handling the viruses themselves. 
40. EKICV 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 percent (w/w) of the total DNA tn 
the preparation, provided that it was veri- 
fied 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 lO’-fold when a nonre- 
petitive gene !rom 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 (v). Le„ "Transfer of a drug resis- 
tance trait to microorganisms that are not 
known to acquire tt naturally if such acqui- 
sition could compromise the use of a drug to 
control disease agents in human or veteri- 
nary medicine or agriculture " 
44. Because this work will be done almost 
exclusively in tissue culture cells, which 
hare no capacity to’- 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 DNA's from a phys- 
ically contained laboratory is carnage by an 
infected human. Thus the vector with an in- 
serted DNA segment should have IKtle 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 folio* mg 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 nonpermisslve cells ( l.e.. cells which do 
not produce Infectious virus panicles), this 
is not a requirement. 
(II) It should be derived from a vims 
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 (Le.. props gatlan of the recombinant 
DNA as a virus must be dependent upon the 
presence of a oomplemetitirg helper 
renome). In almost an eases this oondition 
would be achieved automatically by the ma- 
nipulations used to eonetruct 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 poss:ble to introduce 
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 biloglcally 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 sited at which DNA synthesis origi- 
nates and terminates. 
(ii) the sites that are cleaved by restriction 
endonucleases. 
(iil) the template regions for the major 
gene product. 
If possible the helper virus genome 
should: 
(i) be integrated into the genome of a 
stable Une of host cells <a situation that 
would effectively limit the growth of the 
vector recombinant to such cell lines) or 
FEDERAL REGISTER. VOL 43. NO. 144 — FRIDAY, JULY 2R, 197* 
[ 49 ] 
