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Many self-cloning experiments with agriculturally significant and other gram- 
negative bacteria could be more readily and safely carried out by using well-char- 
acterized E. col i plasmid vectors. Some organisms may not have usable vectors. 
It will be a waste of time and money if researchers choose to develop a new 
"natural" vector for every new bacterium only to avoid regulations. 
(2) La Jolla Group Recommendation IV . The list of "exchangers" among gram- 
negative bacteria should not be restricted to "chromosomal exchangers" but 
should include organisms that exchange any DNA (exceptions for CDC classes 3 and 
4). 
This can be justified easily on the grounds that in vivo methods are appli- 
cable to most, if not all, members of this group. Furthermore, in vitro 
genetic manipulations in these bacteria do not pose substantially greater bio- 
hazard than in vivo genetic manipulations. In many cases, the in vitro methods 
can be judged as safer than the in vivo . 
For example, many researchers use the P-plasmids as conjugative vehicles in a 
variety of bacteria. These plasmids transfer to almost any gram negative species 
and recombine with bacterial chromosomes and other plasmid repl icons. P-prime 
derivatives of these plasmids carrying segments of chromosomal material from 
diverse organisms have been obtained in vivo . Recently, P-plasmid -Mu phage 
cointegrates have been used to induce the transposition of bacterial chromosomal 
segments into P-plasmids. Secondary transmission of these conjugative P-prime 
elements cannot reasonably be judged as less probable than of in vitro recombinant 
plasmids the vectors of which are non-conjugative and have a much more restricted 
host range than the P-plasmids. Furthermore, conjugation allows for the transfer 
of extended regions of "donor" DMA, whereas individual in vitro recombinant plasmids 
are likely to carry only short segments of "donor" DNA. Formation of plasmid- 
primes in vivo is generally mediated by IS sequences. These subsequently promote 
recombination and integration. DNA fragments cloned in vitro will not generally 
contain neighboring IS sequences from the donor chromosomes. With this perspective 
I find it hard to justify the fact that NIH should, for example, regulate the cloning 
of Rhizobium DNA in E_. col i when isolation and handling of RP4-primesis rightly left 
up to the individual investigator. 
If political expediency at this moment or other arguments dictate the approval 
of a restrictive "chromosome exchanger" list rather than the liberal "plasmid ex- 
changer" one, plasmid-prime mediated transmission among gram negative bacteria 
should be regarded as equivalent to other forms of chromosomal gene transmission. 
(3) Definition of HV-1 Systems . The revised guidelines (Fed. Reg. Sept. 21, 1977, 
Part III, p. 49600, column 2, paragraph 2 "other procaryotes"), explicitly pro- 
hibit conjugative plasmids and generalized transducing phages in EK-1 and HV-1 
systems. It is implicit that these elements shall not be introduced subsequent to 
cloning. Literally interpreted, this precludes, for example, vector host range 
tests in nontransformabl e species of bacteria. It also prevents plasmid-promoted 
mobilization to introduce recombinant molecules from a primary cloning host back to 
the original DNA source organism or its mutants. This will effectively preclude 
compl ementaTion tests for traits not expressed in the cloning host, such as plant- 
bacterium interactions in symboisis or pathogenicity. Even in transformable species, 
[Appendix A — 280] 
