33176 
NOTICES 
H.D. a. HV-1 (p. 49600). 
This committee proposes that the 
Recombinant Advisory Committee 
consider allowing the construction of 
modified HV-1 systems with conjuga- 
tion proficient plasmids In addition to 
other recombinant molecules of pro- 
karyotic origin under one step higher 
physical containment providing that 
all the DNA segments In the cell are 
derived from organisms which ex- 
change DNA by natural physio- Lower 
Eukaryotes (p. 9601 Xe)2. Insert the 
words — 
The remainder of the species in this 
class. Including plant pathogenic or 
symbiotic fungi that do not produce 
potent toxins: P2 + EK 1 or PI + EK2. 
Rationale: There Is no demonstrable 
risk either to man or plants from clon- 
ing such DNA In E. coll Also the pres- 
ent wording which refers to disease 
causing microorganisms could be Inter- 
preted to call for an unreasonably 
high containment level for these plant 
pathogens. 
(f) Plants, (p. 49601). Delete the 
words “carries a known pathogenic 
micro-organism or" 
Rationale: The risk to man or plants 
from DNA of a plant pathogen Is not 
comparable to the risk of cloning DNA 
which codes for a potent polypeptide 
toxin. W% have covered this risk else- 
where. 
II. D. Biological containment— Host 
vector systems, a.2. Other prokaryotes 
(p. 49600). 
We endorse the La Jolla Working 
Oroup Draft: (Insert III-3). "Experi- 
ments that are exempt from these 
guidelines". In the event that sections 
(ill) and (Iv) are not adopted we pro- 
pose the following: 
Self-cloning of bacterial plant patho- 
gens and symbionts: 
(I) The use of an Indigenous plasmid 
or bacteriophage shall be exempt from 
the guidelines. 
(II) The use of a foreign vector (a 
non -Indigenous plasmid or bacteri- 
ophage) from an organism which ex- 
changes DNA by natural physiological 
processes shall require P2 contain- 
ment. 
Rationale: Many sslf-clonlng experi- 
ments with agriculturally significant 
gram-negative bacteria could be more 
readily and safely carried out by using 
well characterized E. coll plasmid vec- 
tors. Some plants pathogens and sym- 
bionts Bacterlol.. 22: 138) Pseudo- 
monaj aeruginosa. a pathogen of man. 
conversely. Has been reported to cause 
a leaf -spot of tobacco but Is considered 
a minor and inconsequential pathogen 
of plants (Cho, J. J„ Schroth. M.. 
Mason. M. N, Komlno. S. D. and 
Green. 8. K.. 1975 Phytopath. 65:425- 
431). These three bacteria should be 
governed by regulations applicable to 
human pathogens. 
In our opinion It is a mistake to 
equate prokaryotic plant pathogens 
with Class 2 human pathogens as Is 
done in the revised guidelines. The 
minimum containment level for those 
that have been extensively character- 
ized as to pathogenic and other prop- 
erties should be consistent with that 
adopted for other prokaryotes namely. 
PI + EK2 or P2-f EK1. 
Ill BltXlXd) Viruses of plants (p. 
49602). Change to P2 > Ekl or Pl+Ek2. 
Rationale: Because of their fastidi- 
ous modes of transmission and restric- 
tive host ranges. DNA plant viruses 
were considered to present a minimal 
risk to animals or agriculture when 
used in shotgun experiments with the 
E. coll K-12 host vector systems. 
Ill B 3b Pesticide baculovlruses (p. 
49603). 
(1) Remove sentences "Two viruses 
are presently registered • * • tussock 
moth". 
Rationale: Footnote No. 7 In the 
September. 1977. draft describes the 
baculovirus pesticides that have been 
registered to date. The second sen- 
tence of the September. 1977, draft Is 
therefore repetitive. Also. It should be 
made clear that any baculovirus that 
Is registered by the EPA may be used 
as a vector since EPA registration Is an 
ongoing process and other baculovlr- 
uses may eventually be registered 
which could be more; useful for vector 
work. 
(2) Remove the sentence "However, 
much still needs to be learned” and re- 
write the final sentence of first para- 
graph to read "However. Information 
is needed on the nature of the host 
range specificity, particularly the in- 
fectlvity and persistence of the viral 
DNA In Invertebrate and vertebrate 
cell cultures." 
Rationale: The original sentence. 
"However much still needs to be 
learned." introduces ambiguity. The 
background Information that was 
agreed to be essential In 1977 was in- 
formation on the host specificity, par- 
ticularly lnfectlvtty of viral DNA in 
vertebrate cell cultures. 
(3) Substitute for the last paragraph 
In this section: When such background 
information is available, and if it con- 
firms the narrow host range specifici- 
ty. a baculovirus vector may be used 
for cloning DNA segments derived 
from the host insect, from another En- 
vironmental Protection Agency regis- 
tered baculovirus. from an EK1 bacte- 
rium of from DNAs cloned In an EK1 
bacterium (with the exception of any 
cloned DNA derived from an animal 
virus other than an EPA registered ba- 
culovirus), using P2 physical contain- 
ment. Cloning of other classes of DNA 
Is not envisioned for the exploratory 
phases of this work, but may be con- 
sidered on a case-by-case basis In the 
future. 
Rationale: The terra "EK1 bacte- 
rium" was originally meant to Include 
any DNA cloned in an EK1 bacterium, 
not simply E. colt K-12 DNA. Of par- 
ticular Interest In this category are Le- 
pidopteran genes already cloned in E. 
coll K-12 such as the B. mori silk gene 
and the chorion genes of A. polvphe- 
mw. Also. It would be of Interest to 
extract baculovirus DNA cloned In 
EK 1 with a plasmid vector and test ln- 
fectivlty and/or effects in insect cell 
cultures. Such experimentation is also 
relevant to safety assessment of EK1 
hosts. 
Rationale: The revised guidelines ex- 
plicitly prohibit conjugatlve plasmids 
and generalized transducing phages In 
EK-1 and HV-1 Systems. It is implicit 
that these elements shall not be intro- 
duced subsequent to cloning. This pre- 
cludes host range tests unless they are 
carried out under more stringent phys- 
ical containment. It also prevents plas- 
mid promoted mobilization to Intro- 
duce recombinant molecules back to 
the original DNA source organism or 
Its mutants. Plant pathogens and Rhl- 
zobla are often non-transformable. 
This will effectively preclude comple- 
mentation tests for traits not ex- 
pressed In the cloning host such as 
plant pathogenicity in EK hosts. In 
effect, this creates a dilemma In the 
proposed revisions of the drafted 
guidelines self -cloning in plasmids will 
be excluded even though the recom- 
binant DNA can be mobilized out of 
the host strain Into other prokaryotes. 
On the other hand. DNA cloned from 
the donor prokaryotes into a different 
recipient prokaryote cannot be mobi- 
lized by a conjugatlve plasmid back 
Into the original donor, despite the 
fact that It Is receiving Its own DNA 
by this procedure. If the “exchanger" 
list Is based on plasmid exchange 
these arguments are Irrelevant. 
We have listed In table 2 our sugges- 
tions for prokaryotic exchangers that 
are either plant pathogens or sym- 
bionts (Rhlzobium) together with evi- 
dence for such exchange. 
FiPCIAl IKUSTII. VOC 43. MO. 144— HUDAY, XX Y 23. 1971 
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