33164 
NOTICES 
L corresponds to a containment level 
approximately equivalent to P2. 
Si corresponds to a containment 
level approximately equivalent to P3. 
Prokaryotic sequences that are 
known not to contain toxigenic genes 
may be cloned In L conditions: other- 
wise M conditions should be used. 
+ Before this viral DNA can be used 
as a vector, further information is re- 
quired about its host-range, particular- 
ly Its interactions with cultured pri- 
mate cells. 
CbC these experiments should be as- 
sessed by the appropriate committees 
on a Case by Case" (CbC) basis. 
The group recommended that fur- 
ther work on the biological properties 
of recombinants formed between the 
genomes of two animal viruses should 
be carried out as a matter of some im- 
portance. The workshop participants 
discussed the possibility that new viral 
agents might be created with novel 
biological properties (e.g. host range, 
tissue troplsm. pathogenicity) not 
found in either parent. Several model 
experiments were proposed to test this 
possibility. The group recommended 
that research In this area proceed cau- 
tiously with each case being consid- 
ered on its Individual merits and even 
if the model experiments suggest little 
cause for concern, continued careful 
surveillance of new recombinants 
should be maintained. 
1. Vertebrate host-vector systems in 
icAicA recombinant DNAs are used to 
transform cells. In these types of ex- 
periments viral DNAs carrying a for- 
eign DNA segment will be used to 
transform cells in culture and in the 
process. Integrate the recombinant 
DNA into the host cell chromosome. 
Some transformation systems are non- 
permissive for progeny virus produc- 
tion and pose no possibility of produc- 
ing laboratory Infections. Other trans- 
formation systems are semi -permissive 
and. in addition to the appearance of 
transformed cells, allow the produc- 
tion of low titers of Infectious virus. 
When recombinant DNAs are used 
to transform cells which do not yield 
significant quantities of Infectious 
virus ( e.g SV40 in murine cells, po- 
lyoma virus in rat or hamster cells, 
adenovirus 2 and 5 in rat cells) L con- 
ditions are generally sufficient; for vir- 
uses which do not infect humans, 
there need be no requirement that the 
vector be disabled. Si conditions 
should be used if the system is semi- 
permissive <Le.. virus is produced, in 
low titer) and the virus is capable of 
infecting humans (e g SV40 in human 
cell cultures). It was agreed that the 
DNA to be cloned must not be derived 
from another animal virus. 
The use of viral genes as selective 
markers offers exciting and important 
possibilities for experiments involving 
cloning in eukaryotic cells. Possibly 
the best of such markers would be the 
herpes simplex virus thymidine kinase 
gene We recommend that special em- 
phasis be given to cloning in prokaryo- 
tic systems a small fragment ( < 5KB) 
of herpes viral DNA which contains 
the sequences of this gene. Once avail- 
able. the purified segment could be li- 
gated to any chosen piece of DNA and 
cells transformed by the recombinant 
could easily be selected by virtue of 
the presence of thymidine kinase. 
3. Baculoviruses as vectors. The 
group discussed the use of baculovir 
uses (large DNA -con taming insect vir 
uses) for cloning genes In Invertebrate 
cells and concluded that our knowl- 
edge of the molecular and cellular bi- 
ology of these viruses Is too limi ted to 
allow any general recommendations. 
Proposals to use these viruses should 
be considered case by case. The group 
also recommended, however, that be- 
cause of the potential exploitation of 
these viruses as biological pesticides 
(two have already been licensed for 
this purpose) and as vectors In recom- 
binant DNA experimentation, high 
priority should be given to studies of 
their basic virology, genetics, and mo- 
lecular biology. 
4. Cauliflower Mosaic Virus as a 
vector for cloning genes in plant cells. 
The only known plant viruses which 
could serve as vectors for cloning 
genes in plants and plant cell proto- 
plasts are Cauliflower Mosaic Virus 
(CaMV) and its close relatives, which 
have relaxed circular double stranded 
DNA genomes with a molecular weight 
of 5x10 *. The genomes of these viruses 
are not known to integrate into host 
chromosomes, or to pick up cellular 
genes. CaMV is spread in nature by 
aphids, in which it survives for a few 
hours. Spontaneous mutants of CaMV 
that are not transmitted by aphids 
arise frequently; these mutants fail to 
make a transmission factor essential 
for aphid transmission. 
The viruses in the CaMV group have 
narrow host ranges and are relatively 
difficult to transmit mechanically to 
plants. For this reason, they are most 
unlikely to be accidentally transmitted 
from spillage of purified preparations 
of the virus. 
The workshop participants recom 
mended that for use as a vector with 
Intact plants, a strain should be select 
ed which is not transmitted by aphids 
The ability to produce local lesions in 
an appropriate host would be an ad- 
vantage in maintaining the integrity 
of the strain. The plants should be 
grown in either a greenhouse or plant 
growth cabinet which is insect -proof 
Soil, plant pots and unwanted infected 
plant materials should be removed 
from the greenhouse or cabinet in 
sealed insect proof containers and 
sterilized. It is not necessary to steril- 
ize run-off water from the infected 
plants as this is not a plausible route 
for secondary infections. Infected 
plant materials to be used for further 
research, which have to be removed 
from the greenhouse or cabinet, 
should be maintained under insect 
proof conditions. These measures pro- 
vide an entirely adequate degree of 
containment and are similar to those 
required in many countries for li- 
censed handling of "exotic" plant vir- 
uses. 
CaMV or its DNA may also be useful 
as a vector to introduce genes into 
plant protoplasts. The fragility of 
plant protoplasts combined with the 
properties of CaMV mentioned above 
provides adequate safety. Since the 
group envisaged no risks to the envi 
ronment from use of the CaMV proto- 
plast system, no special containment 
was recommended. 
FHKftAl. (COUTH. VOL 43. NO. 14*— HIOAY, JUIY 2*. 1971 
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