13 
IV. CRITIQUE OF "CONSTRUCTION, PROPERTIES AND TESTING OF >1776" 
We divide our criticism of the "Construction, Properties and 
Testing of >(1776" into two sets of comments-- those dealing with the 
experiments which have been reported and those suggesting some experiments 
which have yet to be done to satisfy the criteria required of an EK-2 
host-vector system according to the NI11 Proposed Guidelines for Research 
Involving Recombinant DNA Molecules (January 1976). 
A. EXPERIMENTS REPORTED 
Strain construction ; 
Si. The construction of Xl?76 from XJ-276 (W1455) involved the use 
of three major techniques for the introduction or removal of markers: 
conjugation, transduction by P1L4, and nitrous acid mutagenesis. During 
this construction isogenecity to the parent strain was not maintained, 
so that comparisons between the phenotypes of the two strains cannot be 
based only on the marker differences intentionally introduced. 
§2. Several markers introduced into this strain are uncharacter- 
ized. This class includes: 
a) the reverted thermosensitive lesion cotransduced into xl678 
with the dapD 8 tonA 53 marker pair, 
b) the nalidixic acid resistance allele, 
c) the unexpected rfb-2 "allele"/phcnotype obtained concommittant 
with the removal of the his -53 allele, and 
d) the thermosensitive mutation unintentionally cotrnnsduced with 
the presumed thyA 57 allele. 
§3. The direct use of nitrous acid to induce anaerobic chlorate 
resistance in X1846 to produce x^849 may be unwise, since nitrous acid, 
a generalized mutagenic agent, may introduce additional uncharacterized 
[Appendix C — 201] 
