Federal Regtolar / Vol. 40. No. 54 / Friday. March 20. 1961 / Notices 
17997 
1. Coattlnnwnt Cooditioa* for Clooina of 
T«dea Coom la Orsaalam* Othor Than E. 
CnllK-U 
lUsuMK Involvini tha clonine of genet 
coding for toxina for veriebralet In hoel- 
vecior tytlemt other than £ co// K-12 will be 
evaluated by OROA. which will contult with 
the od hoc working group on toxina. (See 
Section IV-&-j-tH3Hj)) 
C Section V, Footnote 2A would be 
Bodifled to delete the words: “ 'toxins 
potent for vertebrates' (Section l-D-2)" 
D. A new Section rV-E-l-tH3Hl) 
would be added ai follows: ‘TV-E-l-t>- 
(SHU- Adding new entries to the list of 
toxins for vertebrates. (See Appendix 
C.)" 
E. A new Section lV-E-l-b-(3HJ) 
would be added as follows: "IV-E-l-t>- 
(9HJ)- Approving the cloning of toxin 
genes in host-vector systems other than 
£ co// K-12. (See Appendix C.)” 
DC. Qonlng and Expraaaioo of DNA 
Cndhig for Diphtheria Toxin 
Dr. John Murphy of Harvard 
University has proposed that the 3.0 kb 
Bam restriction fragment of 
Corynophage Beta which carries the 
stnictural gene of diphtheria toxin be 
clotMd in £ co// K-12. 
He advances the following arguments 
as to why these experiments should be 
permitted: 
(1) Studies of diphtheria toxin- 
pr^ucing organisms would yield 
valuable data for risk assessment. 
(2) To determine the localisation of 
di^theria toxin in £ co// and Its 
poeaible secretion by £ co//. 
(3) Mutagenesis of the lox gene would 
be uMd to elucidate (a) diphtheria toxin 
tatarection with the eukaryotic cell toxin 
receptor, and (b) the mechanism of 
fragment A traiulocation Into the 
eui^ryotlc cell cytosol. 
X. Raqueat for Panniaeioa to Ckme the 
Vlfrfio Choiorae Entsrotoxin Geoe in E. 
(>dlK-U. 
Dr. |. ]. Mekalanos of Harvard 
Medical School hes requested an 
exemption from Section 1-0-2 of the 
Guidelines This section prohibits the 
‘formation of recombinant DNAs 
containing genes for the bioaynthesis of 
toxiju potent for vertebrates.” 
2-A200JJ OII3(0»XIS-MAIt-ll-IS:14:39) 
Dr. Mekalanos requests the exemption 
based on the fact that naturally 
occurring transmissible plasmids, which 
encode a heat-labile enterotoxin (LT). 
exist in £ coli. LT has recently been 
shown to share a high degree of 
structural, antigenic and DNA sequence 
homology with cholera enterotoxin. In 
addition, both the LT and cholera 
enlerotoxins are composed of two 
subunits (A and B) both of which are 
required for toxicity. 
Dr. Mekalanos requests consideration 
and approval of the appropriate 
containment level for the experiments 
which will be performed in three stages, 
i.e.. the cloning in £ coU K-12 of specific 
cholera DNA restriction enzyme 
fragments known to contain sequences 
homologous to: (1) The LT A subunit 
gene. (2) the LT B subunit gene. (3) the 
LT A and B subunit genes. 
Since stage 3 experiments are 
anticipated to produce recombinant 
DNA molecules which might express 
active cholera enterotoxin in £ coH K- 
12 hosts, these experiments might be 
conducted at more stringent levels of 
containment than either stage 1 or 2. 
Dr. Mekalanos also proposes to 
perform risk assessment experiments in 
appropriate animal models to evaluate 
the pathogenicity of £ coH K-12 
recombinant clones constructed during 
the course of this work. 
XI. Request To Clone the Geoebc 
Determinant of the Toxic-Shock 
Syndrome Caused by Staphykxoccus 
Aureus 
Dr. Richard Novick of the Public 
Health Research Institute of the City of 
New York. Inc., requests permission to 
clone in Slaphylococcua aureut the 
genetic determinant of the toxic-shock 
syndrome caused by S. aunui. Dr. 
Novick states that recombinant DNA 
techniques promise the most rapid and 
direct means of unraveling the biology 
of this determinant and of the disease in 
which it is implicated. He requests 
permission to conduct the experiments 
under P2 containment using 5. aunu$ 
strain. RN450. which is non-hemolytic. 
non-pigmented. and lacking any 
detectable prophage. 
XII. Request To Use an E. Coli Strain 
Containing a MU Phage Insertion 
Dr. Daryl Holten of the University of 
California at Riverside, requests 
permission to utilize the E. coli K-10 
strain DF 214 (or derivatives thereof), 
and EK plasmid vectors (e.g.. pBR322. 
pBR325) to clone rat cDNA. Strain DF 
214. a K-12 derivative, contains a Mu 
phage insertion in the phosphoglucose 
isomerase gene. 
XIII. Request To Employ a Conjugative 
Plasmid To Transfer N. Crassa DNA 
Dr. Norman Giles of the University of 
Georgia requests permission to use a 
conjugative plasmid to transfer the QA2 
gene of Neuroapora crassa among £ 
coli K-12 strains. The N. crassa QA2 
gene would be ligated into a derivative 
of the mobilizable plasmid RSF2124. 
Oatad: March 13. 1961. 
Oociald S. Fradiickaoo. 
Director. National Institutes of Health. 
Note.— OMB't "Mandatory Information 
Raqoiraments for Federal Aatittance Program 
Announcements” (43 FR 39592) requires a 
slatemeni concerning the official government 
programs contained in the Catalog of Federal 
Domestic Assistance 
Nonnelly NIH lists in its announcements 
the number and title of affected individual 
programs for the guidance of the public. 
Because the guidance in this notice covers 
not only virtually every NIH program but also 
essentially every federal research program in 
which DNA recombinani molecule tet^niques 
could be used. It has been determined to be 
not cost effective or in the public interest to 
attempt to Hat these programs. Such a list 
would llkaly require several additional pages. 
In addition. NIH could not be certain that 
every federal program would be included as 
many federal agencies, as well as private 
organiutions. both national and 
intemattonal. have elected to follow the NIH 
Cuideliites. In lieu of the Individual program 
listing. NIH Inviles readers to direct 
questions to the information address above 
about whether individual programs listed in 
the Catalog of Federal Domestic Assistance 
are affect^. 
NIH programs are not covered by OMB 
Circular A-95 because they fit the description 
of "programs not considered appropriate" in 
Section 8-(b)-(4) and (5) of that Circular, 
irt Ooc si-sassriM s-is-ei essMi 
BxuMO coot 4i«a-aa-ei 
r«wi 
[77] 
