4 
appeared in the Federal Register . The group could subsequently offer a 
recommendation on experiments involving Carrpy lobacter . 
Or. Ifevine pointed out that investigators in other parts of the world may 
perform the types of experiments requested by Dr. O'Brien under much less 
restrictive conditions. He thought U.S investigators are unfairly shackled by 
differences m rules on the cloning of toxin genes. 
Dr. Oottesman said there are two approaches to dealing with differences in 
rules between countries: (1) the working group may suggest the NIH Guidelines 
be modified; and (2) the investigator may request more generic approvals. 
Dr. Gottesman suggested the working group proceed by voting on those portions 
of the request which were published in the August 19, 1985, Federal Register 
and subsequently prepose ard vote on other actions the working group deems 
reasonable . 
Dr. Gottesman asked whether the SLT structural gene is carried on a phage. 
Dr. O'Brien replied that high toxin production m bacteria usually appears to 
be associated with the presence of SLT converting phages such as 93 3J ard 
H1QA/J but not always. A low level of toxin production can consistently be 
detected in the absence of phage, and may be associated with a chromosomal 
gene. Dr. O'Brien hypothesized that the phage gene may be a variant of the 
chrcmoscmal qene. She suggested other variants of the gene might be found in 
other bacterial species. She added that the Shiga toxin gene appears to be a 
chromosomal gene in Shigella dysentenae 1 since the phage cannot be induced . 
Dr. Gottesman asked what is known about the host range of the SLT converting 
thanes. Dr. John Newland of TSUHS said the phages can infect other EL_ coll 
strains; other bacterial families have not been studied to determine whether 
these phaqes w/ill infect members of these families. 
Dr. Gottesman asked how much toxin was produced when the SLT gene under its own 
regulation on a multicopy plasmid was introduced into the EL_ coll host- vector 
system. Dr. O'Rnen replied that a seven fold increase m toxin production 
compared to the standard strain 933 coll 0157:H7 occurs when the SLT gene 
under its own regulation is introduced into the Eh coll host- vector system. 
Dr. O'Brien said the issue of increased SLT production by the EL_ coll host- 
vector system should be kept in perspective. Under optimal laboratory condi- 
tions, Shiqella dysentenae 1 strain 60R produces 10® cytotoxic toxin doses 
per milligram (mg) protein per cell lysate; the standard strain 933 E. coll 
0157:H7 produces 10' cytotoxic doses (CDs) per mg protein under cptimal con- 
ditions. The E. coll host-vector strain carrying the .SET gene on a multiccpy 
plasmid produces 7x10^ CDs per mg protein under cptimal conditions. This 
level of toxin production is still less than that produce! by the Shigella 
dysentenae 1 strain. She added that Shigella flexnen produces approximately 
l.(o CDs pe r mg protein under optimal conditions. The Vibrio cholerae vaccine 
strain produces 10^ CDs per mg protein under optimal conditions. 
[ 308 ] 
