48344 
Federal Register / Vol. 50, No. 22G / Friday. November 22. 1985 / Notices 
DEPARTMENT OF HEALTH AND 
HUMAN SERVICES 
National Institutes of Health 
Recombinant DNA Research; Actions 
Under Guidelines 
agency: National Inslilutes of Health 
(Nil I). PUS, DHHS. 
action: Notice of actions under NIH 
Guidelines for Research Involving 
Recombinant DNA Molecules. 
summary: This notice sets forth four 
actions taken by the Director. NIH. 
under the November 1984 NIH 
Guidelines for Research Involving 
Recombinant DNA Molecules (49 FR 
40268). 
EFFECTIVE date: November 22, 1985. 
FOR FURTHER INFORMATION CONTACT: 
Additional information can be obtained 
from Dr. William J. Cartland, Office of 
Recombinant DNA Activities, National 
Institutes of Health, Bethesda, Maryland 
20892. (301) 496-6051. 
SUPPLEMENTARY INFORMATION: Today 
four actions are being promulgated 
under the NIH Guidelines for Rese&rch 
Involving Recombinant DNA Molecules. 
Three of the proposed actions were 
published for comment in the Federal 
Register of August 19. 1985 (50 FR 
33462), and reviewed and recommended 
for approval by the Recombinant DNA 
Advisory Committee (RAC) at its 
meeting on September 23. 1985. The 
fourth action was published in the 
Federal Register of March 28, 1985 (50 
FR 12456). It was considered and tablea 
at the May 3, 1985, RAC meeting: it was 
rereviewed and recommended for 
approval at the September 25, 1985, RAC 
meeting. 
In accordance with Section IV-C-l-b 
of the NIH Guidelines, these actions 
have been found to comply with the NIH 
Guidelines and to present no significant 
risk to health or the environment. 
Part I of this announcement provides 
background information on the actions. 
Part II provides a summary of the 
actions of the Director, NIH. 
I. Decision on Actions Under NIH 
Guidelines 
A. Request for Permission to Clone 
Shiga-Like Toxin From the Families 
Enterobacteriaceae and Vibrionaceae in 
E. coli K-12 
Dr. Alison O'Brien of the Uniformed 
Services University of the Health 
Sciences (USUHS) had previously (49 FR 
36052) received permission from the 
National Institutes of Health to clone the 
structural gene of the Shiga-like toxin 
(SLT) of Escherichia coli in E. coli K-12. 
Subsequently in a letter of July 26, Dr. 
O'Brien requested permission to clone 
SLT structural genes, defined either by 
nucleotide sequence homology with SLT 
gene probes from E. coli or by antigenic 
cross-reactivity of their gene products 
with purified E. coli SLT. from bacterial 
species classified in the families 
Enterobacteriaceae or Vibrionaceae 
into E. coli K-12 under conditions 
similar to those specified in the previous 
permission. Dr. O'Brien requested 
premission to broaden the scope of 
study of SLT toxin for the following 
reasons: (1) To investigate the potential 
role of SLT in the pathogenicity of 8 
variety of bacteria: (2) to compare the 
SLT genes from various pathogenic 
bacteria; and (3) to facilitate the 
development of an experimental Vibrio 
cholerac vaccine strain deleted for SLT 
genes. 
This proposal was published in the 
August 19, 1985, Federal Register for 
public comment. No cumments were 
received during the comment period. 
On August 16, 1985, the RAC Working 
Group on Toxins considered this 
proposal. A portion of the minutes of tb 
meeting pertaining to this proposal 
reads as follows: 
"Dr. Gottesman said Dr. Alison 
O’Brien of the Uniformed Services 
University of the Health Sciences 
(USUHS) was requesting permission to 
clone SLT structural genes (defined 
either by nucleotide sequence homology 
with SLT gene probes from E. coli or by 
antigenic cross-reactivity of their gene 
products with purified E. coli SLT) from 
bacterial species classified in the 
families Enterobacteriaceae or 
Vibrionaceae according to Bergey s 
Manual of Systematic Bacteriology into 
E. coli K-12 under the conditions 
specified in Federal Register, Volume 49, 
Number 179. Dr. O'Brien had previously 
sought and had obtained permission 
from the NIH to clone SLT from E. coli 
in E. coli K-12 host-vector systems. The 
language of that permission appeared in 
Federal Register 49, Number 179, and 
was incorporated into the NIH 
Guidelines as Appendix F-IV-H 
"Dr. Gottesman said Appendix F-IV- 
H of the NIH Guidelines currently reads. 
‘The intact structural gene(s) of the 
Shiga-like toxin from E. coli may be 
cloned in E. coli K-12 under BL3 + EK". 
containment conditions. 
'E. coli host vector systems expressing 
the Shiga-like toxin gene may be moved 
from BL3 to BL2 containment conditions 
provided that: (1) The amount of toxin 
produced by the modified host-vector 
systems is no greater than that produced 
by the positive control strain 933 E. coli 
015 7:H7. grown and measured under 
optimal conditions; and (2) the cloning 
vehicle is to be an EKl vector preferably 
belonging to the class of poorly 
mobilizable plasmids such as pBR322. 
pBR328. and pBR325. 
‘Nontoxinogenic fragments of the 
Shiga-like toxin structural gene(s) may 
be moved from Bl -3 + EKl to BL2 + F.Kl 
containment conditions or such 
nontoxinogenic fragments may be 
directly cloned in E. coli K-12 under 
BL2 + EK1 conditions provided that the 
E. coli host-vector systems containing 
the fragments do not contain 
overlapping fragments which together 
would encompass the Shiga-like toxin 
structural gene(s).’ " 
. . Dr. O'Brien said she and her 
group had been investigating organisms 
for the presence of SLT. SLT is defined 
as being cytotoxic to cells and 
antigenically cross-reactive with 
purified E. coli SLT. SLT has been 
identified in members of the families 
Enterobacteriaceae and the 
Vibrionaceae. and she was now 
requesting permission to clone SLT 
genes from the Vibrionaceae and the 
Enterobacteriaceae in E. coli K-12. The 
major goal of this work is to develop a 
cholera toxin vaccine. 
“Dr. O'Brien suggested SLT may be,a 
common property of these bacteria; in 
organisms such as Shigella dysenteriae 
1, SLT may play a role in virulence when 
a large amount of the protein is 
produced and other virulence factors are 
present. 
"Dr. O'Brien said SLT has also been 
recently identified in one strain of 
Campylobacter and two strains of 
Aeromonas. The Aeromonas are 
classified as Vibrionaceae. The 
Campylobacter had at one time been 
classified by Bergey’s Manual as 
Vibrionaceae but had recently been 
reclassified as an orphan species. 
“Dr. O'Brien pointed out that a 
number of Campylobacter 
characteristics are common to the 
Vibrionaceae. She asked whether she 
might receive permission to clone SLT 
genes from Campylobacter. 
“Dr. Milewgki said the August 19, 
1985, Federal Register announcement (50 
FR 33482) published 30 days before the 
RAC meeting and describing the 
proposal stated that Dr. O’Brien 
requested permission to clone the'SLT 
gene from members of the families 
Enterbacteriaceae and Vibrionaceae 
. . . She suggested the working group 
first vote on the proposal as it appeared 
in the Federal Register. The group could 
subsequently offer a recommendation on 
experiments involving Campylobacter. 
“Dr. Levine pointed out that 
investigation in other parts of the world 
may perform the types of experiments 
[526] 
