Federal Register / Vol. 59, No. 127 / Tuesday, July 5, 1994 / Notices 
34513 
Appendix C-Vl-D. As classified in 
the Third Report of the International 
Committee on Taxonomy of Viruses; 
Classification and Nomenclature of 
Viruses, R.E.F. Matthews (ed.), 
Inter\'irology 12 (129-296), 1979. 
Appendix C-VI-E. i.e., the total of all 
genomes within a Family shall not 
exceed one-half of the genome. 
Appendix D. Major Actions Taken 
Under the NIH Guidelines 
Under Section IV-C-l-b-(l), the NIH 
Director may take certain actions with 
regard to the NIH Guidelines after the 
issues have been considered by the 
RAC. An updated list of these actions 
are available from the Office of 
Recombinant DNA Activities, National 
Institutes of Health, Building 31, Room 
4B11. Bethesda, Maryland 20092, (301) 
496-9838. 
Appendix E. Certified Host-Vcjctor 
Systems (see Appendix I) 
While many experiments using 
Escherichia coli K-12, Saccharon^yces 
cerevisiae, and Bacillus subtilis are 
currently exempt from the NIH 
Guidelines under Section III-E, some 
derivatives of these host-vector systems 
were previously classified as Host- 
Vector 1 Systems or Host-Vector 2 
Systems. A listing of those systems 
follows; 
Appendix E-1. Bacillus subtilis 
Appendix E-l-A. Bacillus subtilis Host- 
Vector 1 Systems 
The following plasmids are accepted 
as the vector components of certified B. 
subtilis systems: pUBllO, pCl94, 
pSl94, pSA2100, pEl94. pTl27, 
pUBll2, pC221, pC223,-and pABl24. B. 
subtilis strains RUB 331 and BGSC lS53 
have been certified as the host 
component of Host-Vector 1 systems 
based on these plasmids. 
Appendix E-I-B. Bacillus Subtilis Host- 
Vector 2 Systems 
The asporogenic mutant derivative of 
Bacillus subtilis, ASB 298, with tlie 
following plasmids as the vector 
component; pUBllO, pCl94, pSl94, 
pSA2l00, pEl94, pTl27, pUBll2, 
pC221, pC223, and pABl24. 
Appendix E-ll. Saccharomyces 
Cerevisiae 
Appendix E-Il-A. Saccharomyces 
Cerevisiae Host-Vettor 2 Systems 
The following sterile strains of 
Saccharomyces cerevisiae, all of which 
have the ste-VC9 mutation, SHYl, 
SHY2, SHY3, and SHY4. The following 
plasmids are certified for use; Ylpl, 
YEp2, YEp4, YIp5, YEp6, YRp7, YEp20, 
YEp21, YEp24, Ylp25, YIp26, YIp27, 
YIp28, Ylp29, YIp30, Ylp31, Ylp32, and 
YIp33. 
Appendix E-III. Escherichia coli 
Appendix E-IIl-A. Escherichia coli (EK2) 
Plasmid Systems 
The Escherichia coli K-1 2 strain chi- 
1776. The following plasmids are 
certified for use; pSClOl, pMB9, 
pBR313, pBR322, pDH24, pBR325, 
pBR327, pGLlOl, and pHBl. The 
following Escherichia coli/S. cerevisiae 
hybrid plasmids are certified as EK2 
vectors when used in Escherichia coli 
chi-1776 or in the sterile yeast strains, 
SHYl, SHY2, SHY3, and SHY4; YIpI, 
YEp2, YEp4, Ylp5, YEp6, YRp7, YEp20, 
\T:p21, YEP24, Ylp25, YIp26, Ylp27, 
YIp28, TTp29, YIp30, YIp31, YIp32, and 
Ylp33. 
Appendix E-llI-B. Escherichia coli (EK2) 
Bacteriophage Systems 
The following are certified EK2 
systems based on bacteriophage lambda; 
Vector 
Host 
Xgt WESkB' 
DPSOsupF 
Xgt l^'ESXB* 
DPSOsupF 
Xgt ZJ virXB' 
Escherichia coli K-12 
XgtALO-XB 
DPSOsupF 
Charon 3A 
DPSO or DPSOsupF 
Charon 4A 
DPSO or DPSOst^ 
Charon 16A 
DPSO or DPSOsupF 
Charon 21 A 
DPSOsupF 
Charon 23A 
DPSO or DPSOsupE 
Charon 24A 
DPSO or DPSOsupF 
Escherichia coli K-12 strains chi-2447 
and chi-2281 are certified for use with 
lambda vectors that are certified for use 
with strain DP50 or DPSOsupF provided 
that the su-strain not be used as a 
propagation host. 
Appendix E-IV. Neurospora crassa 
Appendix E-IV-A. Neurospora crassa 
Host-Vector 1 Systems 
The following specified strains of 
Neurospora crassa which have been 
modified to prevent aerial dispersion: 
Ini (inositolless) strains 37102, 37401, 
46316, 64001, and 89001. Csp-1 strain 
UCLA37 and csp-2 strains FS 590, 
UCLAlOl (these are conidial separation 
mutants). 
Eas strain UCLAlOl (an "easily 
wettable" mutant). 
Appendix E-V. Streptomyces 
Appendix E-V-A. Streptomyces Host- 
Vector 1 Systems 
The following Streptomyces species: 
Streptomyces coelicolor, S. lividans, S. 
parvulus, and S. griseus. The following 
arc accepted as vector components of 
certified Streptomyces Host-Voc;tor 1 
systems; Streptomyces plasmids SCP2, 
SLPl.2, pIJlOl, actinophage phi C31, 
and their derivatives. 
Appendix E-Vl. Pseudomonas Putida 
Appendix E-Vl-A. Pseudomonas putida 
Host- Vector 1 Systems 
Pseudomonas putida strains KT2440 
with plasmid vectors pKT262, pKT263, 
and pKT264. 
Appendix F. Containment Conditions 
for Cloning of Genes Coding for the 
Biosynthesis of Molecules Toxic for 
Vertebrates 
Appendix F-I. General Information 
Appendix F specifies the containment 
to be used for the deliberate cloning of 
genes coding for the biosynthesis of 
molecules toxic for vertebrates. The 
cloning of genes coding for molecules 
toxic for vertebrates that have an LDso 
of <100 nanograms per kilograms body 
weight (e.g., microbial toxins such as 
the botulinum toxins, tetanus toxin, 
diphtheria toxin. Shigella dysenteriae 
neurotoxin) are covered under Section 
IIl-B -1 and require Institutional 
Biosafety Committee and NIH/ORDA 
approval before initiation. No specific 
restrictions shall apply to the cloning of 
genes if the protein specified by the 
gene has an LD 50 >100 micrograms per 
kilograms of body weight. Experiments 
involving genes coding for toxin 
molecules with an LDso of <100 
micrograms per kilograms and >100 
nanograms per kilograms body weight 
require Institutional Biosafety 
Committee approval and registration 
with NIH/ORDA prior to initiating the 
experiments. A list of toxin molecules 
classified as to LD 50 is available from 
NIH/ORDA. Testing procedures for 
determining toxicity of toxin molecules 
not on the list are available from the 
Office of Recombinant DNA Activities, 
National Institutes of Health, Building 
31. Room 4B11, Bethesda, Maryland 
20892, (301) 496-9838. The results of 
such tests shall be forwarded to NIH/ 
ORDA, which will consult with ad hoc 
experts, prior to inclusion of the 
molecules on the list (see Section IV-C- 
l_b_(2)-(e)). 
Appendix F-II. Cloning of Toxin 
Molecule Genes in Escherichia coli K-12 
Appendix F-II-A. Cloning of genes 
coding for molecules toxic for 
vertebrates that have on LD 50 of >100 
nanograms per kilograms and <1000 
nanogroms per kilograms body weight 
(e.g., abrin, Clostridium perfringens 
epsilon toxin) may proceed under 
Biosafety Level (BL) 2 -► EK2 or BL3 + 
EKl containment conditions. 
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