APPENDIX D--24 
tious by rejoining them with separately 
prepared tails. The high Instability of the 
phage would minimize the possibility of 
transfer of the cloned genes into receptive 
bacteria found In nature. Moreover, the pro- 
pagation of the phage can be blocked by 
many conditional mutations, which would 
be designed to block any secondary route of 
escapie, mainly depending on transfer of the 
cloned DNA Into another phage or bacterial 
host. It was recommended further that the 
vector be designed In such a manner as to 
permit easy Insertion and monitoring of the 
foreign DNA and rapid assay of the safety 
features and give a high yield of cloned DNA 
(not less than 10‘* molecules per ml). There 
also was general agreement that host-phage 
systems other than E. coli should be con- 
sidered. especially those restricted to very 
rare and unusual environments. Also, plas- 
mids derived from phage vectors and which 
give very high DNA yields while exhibiting 
safety features, e.g., Xdvcrots, should be 
considered as vehicles for cloned DNA. 
Szybalskl and S. Brenner (Cambridge Uni- 
versity) stressed that research on recom- 
binant DNA molecules may lend Itself to 
very simple and inexpensive mechanical con- 
tainment, e.g., a small sealed glove box. since 
all the vectors that carry such recombinant 
molecules possibly can be both created and 
destroyed in such a box, while development 
of special methods might permit study of 
many properties of the recombinant DNA, 
without ever removing it from the box. 
These safety features were reflected in the 
subsequent presentations. F. Blattner and 
W. Williams (University of Wisconsin) de- 
scribed four specially constructed X-080 
phages which incorporate many of these 
safety features, arid which they named 
Charon phages, for the mythical boatman 
of the river Styx. Some of these highly con- 
tained phages give yields of over IQn par- 
ticles/ml. R. Davis. J. Cameron and K. 
Struhl (Stanford University) found that X 
phages that carry foreign DNA never grow 
as well as the parental vector, which would 
select against their survival in nature. They 
also reported that some eukaryotic genes 
could be expressed in E. coli, partially com- 
pensating for deflclencles in the histidine 
pathway or in polA or Ug functions. These 
investigators surveyed over 1000 strains of 
E. coli isolated in the natural environment 
and did not find a single strain that could 
support propagation of the \vir vector. 
V. Bode (Kansas State University) dis- 
cussed the possibility of growing tall-free X 
heads. Such heads, which are packed wfith 
DNA, are very fragile, unless stored in 0.01 
M putresclne buffer. Head yields close to 
lOn/ml could easily be attained and, when 
required, heads could be quantitatively re- 
joined with separately supplied tails under 
special laboratory conditions. W. Arber, D. 
Scandella and J. Elliott (University of Basel) 
described bacterial host mutants that per- 
mit efficient infection only by phages with a 
full complement of DNA. This permits select- 
ing for vectors that carry lopg fragments of 
foreign DNA. 
K. Matsubara, T. Mukai and Y. Takagl 
(University of Osaka and Kyushu Univer- 
sity) , and G. Hobom and P. Phllllppsen (Uni- 
versity of Freiburg and Stanford University) 
described various defective X plasmids (Xdv) 
that could be used as efficient vectors. Matsu- 
bara has shown that temperature-sensitive 
cro mutations permit obtaining between 
1000 and 30(X) cloned molecules per cell and 
at the same time result In killing of the 
carrier cells at body temperature. The muta- 
tions Ots and Pts were also evaluated as 
safety features. Phllllppsen described many 
new Xdv plasmids created by cutting X DNA 
with HindUl and Baml restriction endonu- 
cleases followed by ligation. The final talk 
by F. Young, G. Wilson and M. Williams 
(University of Rochester) summarized the 
progress on the development of safer Bacillus 
subtilis host mutants and phages, especially 
03, as vectors. New restriction nucleases, 
Bgl-1 and Bgi-2, were also described. 
The morning session on bacteriophage vec- 
tors was followed by a session on plasmid 
vectors that was chaired by D. Helinskl 
(University of California, San Diego). 
Helinskl presented the following properties 
as highly desirable characteristics of a safer 
plasmid vehicle: (a) non-conjugatlve; (b) 
non-mobilizable or poorly mobllizable by a 
conjugative plasmid; (c) possesses little or 
no extraneous genetic information; (d) 
poorly recombines or does not recombine 
with the chromosome of the host cell; (e) 
provides no selective advantage to the host 
celi or the selective property is conditional; 
and (f) possesses mutations that restrict 
its maintenance to a specific host, prevent 
replication at mammalian body temperature 
and/or provide the plasmid with the capa- 
bility of killing any cell to which it might be 
transmitted other than the host cell. V. 
Hershfield (University of California, San 
Diego) described the properties of a variety 
of derivatives of the ColEl plasmid and the 
broad-host range, P-type plasmid, RK2. 
One of the ColEl derivatives, ColEl-frp, con- 
structed in collaboration with C. Yanofsky 
and N. Franklin (Stanford University) pro- 
vides the means to use the tryptophan genes 
of E. coli as a selective marker in trans- 
formation with recombinant DNA in situa- 
tions where it is desirable to avoid antibiotic 
resistance genes. In addition, Hershfield 
described collaborative work with H. Boyer 
that resulted in the development of a mlnl- 
ColEl plasmid and derivatives of this 
plasmid (mini-ColEl-fcan and mlnl-ColEl- 
trp) aa cloning vehicles. Finally, she de- 
scribed the' temperature-sensltlllty prop- 
ertlles of trp and kan derivatives of a tem- 
perature sensitive replication mutant of 
ColEl Isolated by J. Collins (Molecular 
biology Institute, Stockhelm) and hybrid 
ColEl plasmids carrying the EcoRI generated 
Cts fragment of bacteriophage X-trp61. 
J. Carbon (University of California, Santa 
Barbara) described a replica plating method 
that greatly facilitates the detection of 
E. coli clones bearing CcrfEl plasmids. The 
procedure, which utilizes the F, plasmid to 
promote the transfer of a hybrid ColEl 
plasmid to a suitable auxotrophic recipient, 
was successful in identifying clones bearing 
hybrid plasmids carrying a number of differ- 
ent regions of the E. coli chromosome. The 
contributions of A. J. Clark and collabora- 
tors (University of California, Berkeley) 
were relevant to the problem of the mobili- 
zation and subsequent transfer of non-con- 
Jugative plasmids carrying foreign DNA of a 
potentially hazardous nature. Clark de- 
scribed the variations in transmission fre- 
quencies between the nonconjugatlve plas- 
mids pSClOl, pMLSl, pSC138 and a number 
of pSClOl hybrids containing various EcoRI 
fragments of F when the conjugal transfer 
of these plasmids was promoted by several 
different cpnjugatlve plasmids. 
I. C. Gunsalus and collaborators (Univer- 
sity of Illinois) and A. Chakrabarty (Gen- 
eral Electric Research and Development Cen- 
ter) described the properties of .a variety of 
plasmids Isolated from Pseudomonas putida. 
These contributions were followed by a dis- 
cussion on the merits of developing plasmid- 
host systems involving Pseudomonas strains 
that naturally exhibit unusual growth re- 
quirements. Similar studies with plasmids 
isolated from Bacillus megaterium by B. 
Carlton (University of Georgia) from B. 
suhtilis by P. Lovett (University of Mary- 
land) and other naturally occurring Bacillus 
species by W. Goebel and K. Bernhard (Mi- 
crobiology Institute, Wurzburg) were dis- 
cussed and their further development as 
plasmid-host cloning systems was explored. 
It was clear from these presentations that 
considerable progress has been made re- 
cently in the identification and characteri- 
zation of a variety of plasmid elements that 
occur naturally in Pseudomonas and Bacillus 
species. Several of the plasmids described 
show considerable promise as plasmid clon- 
ing .systems involving a host other than 
E. coli. 
A third session on the ecology and epi- 
demiology of vector-host systems was chaired 
by S. Falkow (University of Washington). 
This workshop emerged, in part, from ex- 
pressed fears that microorganisms contain- 
ing cloned fragments of foreign DNA poten- 
tially pose a threat to health or disrupt the 
normal ecological chain in some manner. 
Consequently, this session was devoted to a 
review of currently available information on 
the ecology and epidemiology of E. coli and 
related bacterial species since It was recog- 
nized that E. coli K-12 would be the pro- 
karyotic host most commonly employed In 
the cloning of DNA molecules In the Imme- 
diate future. F. Orskov (Escherichia Refer- 
ence Center, Copenhagen) reviewed the state 
of E. coli serotyplng and what has been 
learned about the dlstrlbtulon of E. coli 
types in health and disease. Only certain 
E. coli types are generally recognized as good 
colonizers of the human gut and such strains 
come from a handful of the 160 well defined 
0 (lipopolysaccharlde) antigen types and in- 
variably posses K (acidic polysaccharide 
capsule) antigens. Some serotypes appar- 
ently have become disseminated worldwide 
and possibly represent the proliferation of a 
bacterial clone because of, as yet unkown, 
selective pressures. In contrast, E. coli K-12 
has no detectable O or K antigens and Is 
considered to be rough. This may accoimt, at 
least In part, for its demonstrable poor abil- 
ity to colonize the human or animal gut. 
However, R. Freter (University of Michigan) 
point out that we still remain largely Ignor- 
ant of the factors which control Intestinal 
E. coli populations. Freter also noted that 
while adherence to the mucosal surface of 
the small intestine is Important in the path- 
ogenesis of E. coli diarrheal disease, the “nor- 
mal” long-lasting sjTnblotic relationship be- 
tween a mammalian host and bacterium is 
established in the cecum and colon. It is In 
these locations that factors come into play 
to determine whether an E. coli strain pass- 
ing through the Intestine will become suc- 
cessfully Implanted or whether It will be 
quickly eliminated In the feces. 
The factors controlling implantation In- 
clude competition for substrates, inhibitors 
and the physiological state of the orgarilsm 
when It reaches the large bowel. For example. 
Ingested E. coli previously grown under usual 
laboratory conditions fare poorly while cells 
of the same strain "pre-adapted” in Eh, pH, 
etc., often colonize well. Freter has developed 
a continuous flow culture model which may 
be useful in studying the mechanisms of 
Implantation. Falkow reviewed the patho- 
genicity of E. coli. E. coli caioses diarrheal 
disease either by- direct Invasion of the bowel 
epithelium or by elaboration of entero- 
■'.oxin(s) . 'While Invasive E. coli appear to owe 
their pathogenicity to a constellation of a 
least five unlinked chromosomal gene clus- 
ters, toxigenic E. coli species generally owe 
their pathogenicity to the possession of two 
species, Ent and K. The introduction of Ent 
and K plasmids may be sufficient to convert 
a normal wild-type E. coli Into a strain now 
capable of causing overt clinical disease. 
However, the Introduction of these plasmlda 
Into E. coli K-12 sublines had no discernible 
effect on their ability to cause disease, al- 
though the K-12 strains could now better 
colonize calves. Despite the observation that 
