33158 
NOTICES 
July 10. 1977. Comment s (peach-col- 
ored book) p.l.” 
Response. The letter to Dr. Fredrick- 
son. July 10 (actually to Dr. Gartland. 
July 5. and received July 10) com- 
ments on the guidelines rather than 
the E1S. All letters received in re- 
sponse to the draft EIS are Included In 
appendix K of the final EIS and are 
responded to on page 113-137 of the 
final EIS. Our response to Comment 5 
above is also applicable to this com- 
ment— l.e., the EIS discusses plants on 
pages 23 and 120. and further discus- 
sion of this issue was held at the 
recent workshop on risk assessment of 
agricultural pathogens, which has led 
to changes in the PRG-NIH. 
Appendix D 
DISCUSSION or THE "LIST OP EXCHANG- 
ERS’" CONSTITUTING APPENDIX A TO THE 
GUIDELINES 
Section I-E-4 of the Guidelines 
stales that certain recombinant DNA 
molecules are exempt from the Guide- 
lines. These are molecules that "con- 
sist entirely of DNA segments from 
different species that exchange DNA 
by known physiological processes, 
though one or more of the segments 
may be a synthetic equivalent. A list 
of such will be prepared and periodi- 
cally revised by the Director. NTH. on 
the recommendation of the Re- com- 
b Inant DNA Advisory Committee, 
after appropriate notice and opportu- 
nity for public comment. Certain 
classes are exempt as of publication of 
these Revised Guidelines. The list Is in 
Appendix A." 
The natural transfer of genes be- 
tween bacteria occurs by transduction 
(bacterial virus- media ted), transforma- 
tion (uptake of Isolated DNA by a bac- 
terial cell), or conjugation (plasmid- 
mediated transfer of genes between 
bacteria, requiring cell-to-cell contact). 
A reasonable generalization is that vir- 
tually all closely related species of bac- 
teria can exchange genes by transduc- 
tion and transformation, the former 
limited by the relatively narrow host- 
range of transducing bacteriophage 
and the latter by the requirement, in 
the case of chromosomal DNA. for ho- 
mology of DNA in most recombination 
event*. Conjugal mating with ex- 
change of DNA can occur between vir- 
tually all Gram-negative bacteria in- 
cluding naturally occurring soil and in- 
testinal species, when mediated by a 
plasmid of broad host-range (for ex- 
ample. the Inc P-1 group plasmids). 
Recently, conjugal mating has also 
been shown to occur between strains 
of certain species of Streptococcus, a 
Gram-positive organism (for example. 
Streptococcus faecalis). To date, how- 
ever. conjugal mating has not been 
demonstrated between Gram-negative 
and Oram-positive bacteria 
The relatedness of different micro- 
bial species can be estimated by deter- 
mining the extent of DNA homology 
between them or by studying the 
properties of different microorganisms 
in genetic crosses. As a general rule, 
organisms that show considerable ho- 
mology of their nucleotide sequences 
under a standard set of experimental 
conditions have the capacity to mutu- 
ally integrate chromosomal genes. For 
example, in the case of the Enterobac- 
tenaceae family of bacteria (Includes 
Escherichia coli K-12). there is both 
extensive DNA-DNA homology (1) and 
chromosomal gene exchange. ( 2 ) with 
a reasonable correlation between the 
degree of DNA-DNA homology and 
the capacity to mutually Integrate 
chromosomal genes. 
Genetic relatedness, as indicated by 
a high level of DNA-DNA homology 
between different microorganisms, is 
not. however, an absolute requirement 
for the exchange of chromosomal 
genes between bacteria In fact, chro- 
mosomal gene transfer among diverse 
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 chro- 
mosomal genes Is promoted 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. In- 
corporate segments of these chromo- 
somes. and are capable of establishing 
themselves along with covalently- 
linked chromosomal genes in a wide 
range of Gram-negative bacteria. ( 3) 
Appendix A to the Guidelines con- 
tains a list of documented cases of 
chromosomal gene exchange between 
a variety of bacteria and E. coli K-12 
where the gene exchange is promoted 
by Inc P-1 or other plasmids. The first 
ten entries in this table are members 
of the Enterobacteriaceae family of 
bacteria The bacterial species in this 
family not only show chromosomal 
gene exchange but. as Indicated above, 
exhibit extensive DNA-DNA homol- 
ogy. References listed at the end of 
this Appendix 14) provide documenta- 
tion for the entries in Appendix A to 
the Guidelines. All entries in Appen- 
dix A exhibit R-prime transfer (R 
plasmid carrying chromosomal genes) 
to E. coli K-12 mediated by the Inc P- 
1 or other plasmids. 
The Recombinant DNA Advisory 
Committee at its April 27-28. 1978, 
meeting proposed three possible alter- 
native lists for consideration by the 
Director. NIH. to become Appendix A 
to the Guidelines. 
The first list with its criteria and en- 
tries was as follows: 
The following organisms of the Entcro- 
bacteriaecae family exhibit chro- mosomal 
DNA relatedness (20 percent or more ho- 
mology of DNA of various pairs tested) and 
genetic recombination or R-prime (R plas- 
mid carrying chromosomal genes) transfer 
to E. coli K-12 mediated by the Inc P-1 
plasmids. 
All species of the following genera: 
1. gjc/lencAia (including E. col: K-12) 
2. Shigellas 
3. Salmonella 
4. Enterobacler 
5. Arizona 
6. Citrobacter 
7. Klebsiellas 
In addition, the following species: 
1. frinnic amylovom 
2. Entnnia dissolvent 
3. Encinia mintpressurahs 
4. Serralia marcescens 
5. Le tinea malonalica 
6. Levi nea amalonatica 
The second list proposed by the 
RAC Included all the members of the 
first list but added additional mem- 
bers. The criteria used are that all the 
members of the list exhibit R-prime 
transfer (R plasmid carrying chromo- 
somal genes) to E. coli K-12 mediated 
by the IncP-1 or other plasmids. This 
is the list which (which certain dele- 
tions) was selected by the Director, 
NIH to become Appendix A to PRG- 
NIH. The references supporting the 
entries to this list are give 14) at the 
end of this Appendix. 
The third list included all the mem- 
bers of the second list but adds addi- 
tional members. This list, with its cri- 
teria and entries, was as follows: 
The following genera and/or species pos- 
sess R plasmids (including R plasmids of the 
IncP-1 group) transferable to E. coli K-12. 
1. All members of the Enterobacteriaceae 
family 
2. Vtbno species (except Vibrio para- hemo- 
ly Ileus) 
3. Pseudomonas species 
4. Rhizobium species 
5. Acinetobacter calcoaceticus 
6. Agrobacterium tume/aciens 
7. Rhodopseudomonas sphaeroides 
8. CaulobacCer crescent us 
9. Proteus species 
10. Achromobacler species 
11. Aeromonas salmon icida 
12. Alcaligenes faecalis 
13. Bordetella branch tsephea 
14. Myzococcus xanthus 
15. Neisseria gonorrhoeas 
16. Pastuerella hemolytica 
17. Pastuerella multocxda 
18. Yersinia species (excludes Y pest is. since 
it is a Class 3 agent) 
19. Xanthomonas species 
In addition, recombinant DNA experi- 
ments between H influenzae and H. parain- 
fluenzae are exempt on the basis of exten- 
sive DNA homology. 
References 
1. Brennei D J. (1977). Characterization 
and Clinical Identification of Entero- bac- 
tcriaceas by DNA Hybridization. In Pro- 
gress in Clinical Pathology 7*71-1 18. 
FtO€«Al lioism. VOt 43. NO. 144— FtlDAY, JULY 28. 1978 
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