60100 
NOTICES 
an absolute requirement for the exchange 
of chromosomal genes between bacteria In 
fact chromosomal gene transfer among di- 
verse members of the Gram-negative group 
of bacteria has been demonstrated where 
the microorganisms involved show little or 
no DNA-DNA homology. In these cases the 
exchange of chromosomal genes is promot- 
ed by a broad- host-range plasmid of the Inc 
P-1 incompatibility type. These plasmids 
mobilize the chromosomes of a wide variety 
of Gram-negative bacteria incorporate seg- 
ments of these chromosomes, and are capa- 
ble of establishing themselves along with co- 
valently linked chromosomal genes in a 
wide range of Gram-negative bacteria (3)" 
In evaluating a pair of organisms for in- 
clusion as nonnovel exchangers, we are 
malting an estimate of the probability that 
the combination of genes might have oc- 
curred naturally. If the combination is not a 
new one. then there should be no special 
hazard in creating such an organism by re- 
combinant DNA technology. Thus we can 
exempt this combination from the Guide- 
lines. 
Any conclusion about exchange between 
organisms involves some extrapolation from 
the experimental data available. We have 
tried, in the discussion below, to make ex- 
plicit these extrapolations and the scientific 
bases for making them. For this purpose, 
the types of criteria which might be taken 
into account in preparing a list of exchang- 
ers are divided Into four categories. The 
first two were those used in constructing 
Appendix A in these final revised guidelines. 
The first three were used In constructing 
the version of Appendix A which appeared 
in the July 28. 1978. proposed revised guide- 
lines. All four were used in constructing a 
list approved by the RAC and described as 
the "third list" In Appendix D to the Jwly 
28. 1978. Environmental Impact Assessment. 
1. Organisms which exchange chromoeo- 
mat genetic information which becomes 
ilaMy integrated into the host chromosome. 
This, the most stringent criterion for ex- 
change. requires significant homology be- 
tween recombining segments. Organisms 
which meet these criteria will therefore be 
closely related by DNA homology measure- 
ments (Ref. 1. 2). In addition, more than 
one mechanism of genetic exchange may be 
found (We.. transduction and plasmid mobili- 
sation), and transfer of many different 
markers may be demonstrated. The major 
extrapolation Involved in this category Is 
the extension of data from one strain to 
others in the same species or genua In the 
Appendix A list, moat of the entries are 
listed as genera (Shigella. Salmonella, etc.), 
while the Pseudomonas aeruginosa species 
is listed. One can generalize from species to 
genus when there is evidence that all mem- 
bers of the genus behave similarly, and 
show extensive DNA homology. In addition. 
In some cases, exchange has been demon- 
strated in many species of the genus. The 
DNA homology test is convincing for Shi- 
gella. but not for Pseudomonas. 
2. Organismj ichicA exchange chromoso- 
mal in/ormation that is not necessarily inte- 
grated into the chromosome of the recipient 
i/or instance, trans/e r ria F or R ). This sort 
of exchange can occur in the absence of ex- 
tensive DNA homology between the organ- 
isms. and requires only that the plasmid and 
Its chromosomal genes be maintained In the 
recipient organism. 
Although two organisms meeting these 
criteria might not be closely related, this 
FEDERA1 
type of exchange is probably the best model 
of a recombinant DNA experiment. In both 
cases, relatively small amounts of genetic in- 
formation are transferred, usually in plas- 
mid form, to an otherwise "foreign" genetic 
background. Therefore, it seems reasonable 
to accept evidence of such exchange as 
grounds for exemption. 
Criteria 3 and 4 deal specifically with Inc 
P-1 plasmids, but the principles can be ex- 
tended to other exchange mechanisms. 
3. Organisms which show evidence of a 
plausible mechanism for exchange ie.g.. R 
formation or evidence of mobilisation of 
chromosomal genes by an Inc P-1 plasmid.) 
In this case, the plasmid itself has been 
shown to move from organism to organism. 
It has been shown to pick up chromosomal 
genes, but the transfer of these chromoso- 
mal genes in interspecies matings has not 
necessarily been demonstrated. 
To endorse these criteria, we must ex- 
trapolate from the transfer within the spe- 
cies to more distantly related organisms. 
For such an extrapolation, one must assume 
that no barriers will exist for R' transfer 
that do not exist for transfer of the plasmid 
itself. In many tested cases, this is clearly 
true: transfer of chromosomal genes incor- 
porated into an R factor is comparable in 
frequency to transfer of the plasmid Itself, 
and the R can be moved to a broad range of 
organisms (4). However, this may not be 
universally true (S). 
4. Organisms whch can receive or donate 
broad host range plasmids. Since these plas- 
mids are known, in many cases, to mobilize 
the chromosome and transfer chomosomal 
genes, such transfer might be expected for 
any organism that receives or donates the 
plasmid. This extrapolation assumes that (i) 
chromosomal pickup is always able to occur 
with these plasmids and <U> transfer of 
chromosomal genes to other species win 
occur (this latter case is the same at that 
analyzed under criterion 3). 
Analysis of the basis for the first extrapo- 
lation would include consideration of the 
numerous cases where such mobilization 
and transfer can be detected (see. for In- 
stance. ref. 5-10) and those few where it 
cannot (11). 
It is my decision that the data supporting 
the use of criteria 3 and 4. while suggestive, 
are not yet compelling enough to warrant 
exemption from the Guidelines for recom- 
binant DNA experiments. As more data are 
accumulated, this conclusion will be careful- 
ly reconsidered. 
77ie Issue of Two-Way Exchange 
The organisms listed in appendix A fit the 
first two criteria described above— i.e., in all 
cases, there is direct evidence of chromoso- 
mal exchange between two species on the 
list, and many show extensive DNA homol- 
ogy as well. In addition, for all the organ- 
isms on this list, exchange can be demon- 
strated in two directions. Further. If organ- 
isms A and B both exchange genetic infor- 
mation (both donate and receive) with a 
third organism— E. coli K-12. for instance- 
then E. coli K-12 can act as a path for the 
DNA of organism A to reach B. and vice 
versa Thus, the requirement for two-way 
exchange allows us to exempt recombinants 
made between A and B. Therefore, we have 
exempted "any recombinant DNA molecules 
that are (1) composed entirely of DNA seg- 
ments from one or more of the organisms 
listed below and (2) to be propagated in any 
of the organisms listed below." 
REGISTER. VOC 43, NO. 247— FRIDAY, DECEMBER 
[ 23 ] 
It would make sense, of course, to have 
one-way lists as well, where cloning exempt 
from the Guidelines would only be allowed 
in the recipient. The creation of such lists 
will be considered by the RAC. 
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