27922 
NOTICES 
Aerobiology. John Wiley & Sons, New York, 
pp. 194-263. 
(18) Grunsteln, M. and D. S. Hogness 
(1975). Colony Hybridization: A Method lor 
the Isolation of Cloned DNAs That Contain 
a Specific Gene. Proc. Nat. Acad. Scl. U.S_A. 
72, 3961-3965. 
(19) Morrow, J. F„ S. N. Cohen, A. C. Y. 
Chang, H. W. Boyer, H. M. Goodman and R. 
B. Helling (1974). Replication and Transcrip- 
tion of Eukaryotic DNA in Escherichia coli. 
Proc. Nat. Acad. Scl. USA 71, 1743-1747. 
(20) Hershfleld, V„ H. W. Boyer, C. Yanof- 
sky, M. A. Lovett and D. R. Hellnskl (1974). 
Plasmid Col El as a Molecular Vehicle for 
Cloning and Amplification of DNA. Proc. Nat. 
Acad. Scl. USA 71, 3455-3459. 
(21) Wensink, P. C., D. J. Finnegan, J. E. 
Donelson, and D. S. Hogness ( 1974) . A Sys- 
tem for Mapping DNA Sequences in the 
Chromosomes of Drosophila melanogaster. 
Cell 3, 315-325. 
(22) Tlmmls, K. F. Cabello and S. N. Cohen 
(1974). Utilization of Tioo Distinct- Modes of 
Replication by a Hybrid Plasmid Constructed 
In Vitro from Separate Replicons. Proc. Nat. 
Acad. Scl. USA 71, 4556-4560. 
(23) Glover, D. M., R. L. White, D. J. Fin- 
negan and D. S. Hogness (1975). Character- 
ization of Six Cloned DNAs from Drosophila 
melanogaster. Including one that Contains 
the Genes for \RNA. Cell 5, 149-155. 
(24) Kedes, L. H„ A. C. Y. Chang, D.- House- 
man and S. N. Cohen (1975) . Isolation of His- 
tone Genes from Un fractionated Sea Urchin 
DNA by Subculture Cloning in E. coli. Nature 
255, 533. 
(25) Tanaka, T. and B. Welsblum (1975). 
Construction of a Colicin El-R Factor Com- 
posite Plasmid In Vitro: Means for Amplifi- 
cation of Deoxyribonucleic Acid. J. Bacteriol. 
121, 354—362. 
(26) Tanaka, T., B. Welsblum, M. Schnoss 
and R. Inman (1975). Construction and 
Characterization of a Chimeric Plasmid Com- 
*posed of DNA from Escherichia coli and 
Drosophila melanogaster. Biochemistry 14, 
2064-2072. 
(27) Thomas, M., J. R. Cameron and R. W. 
Davis (1974). Viable Molecular Hybrids of 
Bacteriophage Lambda and Eukaryotic DNA. 
Proc. Nat. Acad. Scl. USA 71, 4579-4583. 
(28) Murray, N. E. and K. Murray (1974). 
Manipulation of Restriction Targets in Phage 
X to form Receptor Chromosomes for DNA 
Fragments. Nature 251, 476-481. 
(29) Rambach, A. and P. Tlollais (1974). 
Bacteriophage X Having EcoRl Endonuclease 
Sites only in the Non-essential Region of the 
Genome. Proc. Nat. Acad. Sci. USA 71, 3927- 
3930. 
(30) Smith, H. W. (1975). Survival of 
Orally-Administered Escherichia coli K12 in 
the Alimentary Tract of Man. Nature 255, 
600-502. 
(31) Anderson, E. S. (1975). Viability of, 
and Transfer of a Plasmid from Escherichia 
coli K12 in the human intestine. Nature 255, 
502-504. 
(32) Falkow, S. (1975). Unpublished ex- 
periments quoted In Appendix D of the Re- 
port of the Organizing Committee of the 
Asilomar Conference on Recombinant DNA 
Molecules. (P. Berg, D. Baltimore, S. Brenner, 
R. O. Roblln and M. Singer, eds.) submitted 
to the National Academy of Sciences. 
(33) R. Curtis III, personal communica- 
tion. 
(34) Novick, R. P. and S. I. Morse (1967). 
In Vivo Transmission of Drug Resistance 
Factors between Strains of Staphylococcus 
aureus. J. Exp. Med. 125, 45-59. 
(35) Anderson, J. D„ W. A. Gillespie and M. 
H. Richmond. (1974). Chemotherapy and 
Antibiotic Resistance Transfer between En- 
terobacteria in the Human Gastrointestinal 
Tract. J. Med. Microbiol. 6, 461-473. 
(36) Ronald Davis, personal communica- 
tion. 
(37) K. Murray, personal communication; 
W. Szybalskl. personal communication. 
(38) Manly. K. R., E. R. Signer and C. M. 
Raddlng (1969). Nonessential Functions of 
Bacteriophage X. Virology 37 177. 
(39) Gottesman, M. E. and R. A. Welsberg 
(1971). Prophage Insertion and Excision. In 
The . Bacteriophage Lambda (A. D. Hershey, 
ed.) . Cold Spring Harbor Laboratory pp. 113- 
138. 
(40) Shimada, K., R. A. Welsberg and M. E. 
Gottesman (1972). Prophage Lambda at Un- 
usual Chromosomal Locations: I. Location 
of the Secondary Attachment Sites and the 
Properties of the Lysogens. J. Mol. Biol. 63, 
483-503. 
(41) Signer, E. (1969). Plasmid Formation: 
A New Mode of Lysogeny by Phage X. Nature 
223, 158-160. 
(42) Adams, M. H. (1959). Bacteriophages. 
Intersciences Publishers, Inc., New York. 
(43) Jacob, F. and E. L. Wollman. (1956). 
Sur les Processus de Conjugaison et de Re- 
combinasion chez Escherichia coli. I. L’induc- 
tion par Conjugaison ou Induction Zygoti- 
que. Ann. Inst. Pasteur 91, 486-510. 
(44) J. S. Parkinson as cited (p. 8) by 
Hershey, A. D. and W. Dove (1971). Introduc- 
tion to Lambda. In: The Bacteriophage X. 
A. D. Hershey, ed. Cold Spring Harbor Labora- 
tory, New York. 
vn. MEMBERS O? THE RECOMBINANT DNA MOLE- 
CULE PROGRAM ADVISORY COMMITTEE 
Chairman 
Stetten, DeWltt, Jr., M.D., Ph.D., Deputy Di- 
rector for Science, National Institutes of 
Health. 
Vice Chairman 
Jacobs, Leon, Ph.D., Associate Director for 
Collaborative Research, National Institutes 
of Health. 
Adelberg, Edward A., Ph.D., Professor, De- 
partment of Human Genetics, School of 
Medicine, Yale University. 
Chu, Ernest H. Y., Ph.D., Professor, Depart- 
ment of Human Genetics, Medical School, 
University of Michigan. 
Curtiss, Roy, III, Ph.D., Professor, Depart- 
ment of Microbiology, School of Medicine, 
University of Alabama. 
Darnell, James E„ Jr., M.D., Professor, De- 
partment of Molecular Cell Biology, Rocke- 
feller University. 
Hellnskl, Donald R., Ph.D., Professor, De- 
partment of Biology, University of Cali- 
fornia, San Diego. 
Hogness, David S., Ph.D., Professor, Depart- 
ment of Biochemistry, Stanford University. 
Kutter, Elizabeth M„ Ph.D., Member of the 
Faculty, In Biophysics, The Evergreen State 
College. 
Littlefield, John W., M.D., Professor & Chair- 
man, Department of Pediatrics, Children’s 
Medical & Surgical Center, Johns Hopkins 
Hospital. 
Redford, Emmette S., Ph.D., LL.D., Ashbel 
Smith Professor of Government and Public 
Affairs, Lyndon B. Johnson School of Pub- 
lic Affairs, University of Texas at Austin. 
Rowe, Wallace F.. M.D., Chief, Laboratory of 
Viral Diseases, National Institute of Al- 
lergy & Infectious Diseases, National In- 
stitutes of Health. 
Setlow, Jane K., Ph.D., Biologist, Brook- 
haven National Laboratory. 
Splzizen, John, Ph.D., Member and Chair- 
man, Department of Microbiology, Scripps 
Clinic & Research Foundation. 
Szybalskl, Waclaw, D.Sc., Professor of On- 
cology, McArdle Laboratory, University of 
Wisconsin. 
Thomas, Charles A., Jr., PhU., Professor, De- 
partment of Biological Chemistry, Harvard 
Medical School, 
Executive Secretary 
Gartland, William J., Jr., Ph.D., Health Sci- 
entist Administrator, National Institute of 
General Medical Sciences, National Insti- 
tutes of Health. 
Liaison Representatives 
Hedrich, Richard, Ph D., Coordination Pro- 
gram of Science Technology & Human Val- 
,ue, National Endowment for the Humani- 
ties. 
Lewis, Herman W„ PhD., Division of Biologi- 
cal and Medical Sciences, National Science 
Foundation. 
Nightingale, Elena O., Ph.D., Assembly of 
Life Sciences, National Academy of Sci- 
ences. 
Shepherd, George R., Ph.D., Division of Bio- 
medical and Environmental Research, 
Energy Research and Development Ad- 
ministration. 
Appendix A — Statement on the use of 
"Bacillus subtllis” rw recombinant mole- 
cule technology 
Unquestionably, Escherichia coli Is the 
most well characterized unicellular organism. 
Years of basic research have enabled investi- 
gators to develop a well characterized genetic 
map, to obtain detailed knowledge of virulent 
and temperate bacteriophages, and to explore 
the physiology, genetics, and regulation of 
plasmids. More recently, the development of 
DNA-medlated transformation has permitted 
exogenous fragments or molecules of DNA 
to be incorporated into the genome or to 
reside as self-replicatlng units. The dis- 
covery of transformation of Bacillus subtilis 
by Spizlen (1) stimulated the development 
of analternative model system. The purpose 
of this report is to summarize the current 
status of this genetic system and to describe 
the actual and potential vectors and vehicles 
available for recombinant molecule tech- 
nology. 
A. CURRENT KNOWLEGDE OF THE CHROMOSOMAL 
ARCHITECTURE AND MECHANISMS OF GENETIC 
EXCHANGE IN B. “SUBTILLS’’ 
Two mechanisms of genetic exchange have 
been utilized to establish the linkage map of 
B. subtilis, DNA-medlated transformation 
(capable of transferring approximately 1% of 
the genome) and transduction with bacterio- 
phage PBSI (capable of transferring 5-8% of 
the chromosome) . Recent detailed genetic 
studies with PBSI by Lepesant-Kejzlorovi et 
al. (2) have resulted In the development of 
a circular genetic map for this organism. 
The current edition of the map (3) contains 
196 loci. Biophysical analyses have estab- 
lished that the chromosome is circular (4) 
and replicates bidirectionally (5). 
Transformation with purified fragments of 
DNA is a highly efficient process in B. subtilis 
with frequencies of 1 to 4% usually attained 
for any auxothrophic or antibiotic resistance 
markers. Frequencies of approximately 10% 
transformation can be achieved with DNA 
prepared from gently lysed L-forms or pro- 
toplasts (6). These large fragments of DNA 
are readily incorporated by the recipient cell. 
Generalized transduction occurs with bac- 
teriophages SP10 (7), PBSI (8), and SPP1 
(9), while a low frequency of specialized 
transduction has been reported with bac- 
teriophage <fil 05 (10). 
Although transformation is most efficient 
in homologous crosses (B. subtilis Into B. 
subtilis) , it has also been possible to exchange 
DNA among closely related species (11). The 
most extensively studied members of the B. 
subtilis genospecies Include B. licheniformis, 
B. pumilus, B. amyloliquefaciens, and B. 
globigii (refer to reference 12 for a review and 
references 13-15 for examples of this heterol- 
ogous exchange) . This exchange occurs even 
FEDERAL REGISTER, VOL 41, NO. 1 31— WEDNESDAY, JULY 7, 1976 
[ 22 ] 
