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has as yet been certified and since the first 
P4 facility has only recently been certified, 
these experiments were effectively forbid- 
den. The same experiments require signifi- 
cantly lower containment under some Euro- 
pean guidelines. 
(ff) Prof. James Watson, in testimony at 
the December 1977 DAC meeting and in 
print, has sought repentance for his earlier 
activities in support of special precautions 
for recombinant DNA research. ' 
(12) The report, “Science Policy Implica- 
tions of DNA Recombinant Molecule Re- 
search,” March 1978, of the Subcommittee 
on Science, Research, and Technology of 
the Committee on Science and Technology, 
U.S. House of Representatives, says, “The 
burden of proof of safety factors should not 
be borne exclusively by proponents of re- 
combinant DNA research; opponents must 
assume a corresponding burden.” 
(13) Significant differences exist between 
prokaryotes and eukaryotes in the ways pro- 
teins are synthesized under genic direction, 
and these account for limitations on the ap- 
parent success of many recombinant DNA 
experiments to date. A major thrust of cur- 
rent recombinant DNA research is in the di- 
rection of overcoming these differences. 
There is every reason to believe that this re- 
search will succeed. At my invitation. Dr. 
Malcolm Martin of NIH has drawn up this 
brief analysis of the state-of-the-art: 
The potential use of recombinant DNA 
techniques to produce biologically useful 
reagents is predicated on: (a) the faithful 
replication of a segment of foreign DNA in a 
new host cell; (b) the synthesis of messenger 
RNA (mRNA) complementary to the Insert 
ed DNA; and (c) the efficient translation of 
the mRNA into a polypeptide. In nearly all 
cases that have been examined to date, 
DNA, from both eukaryotic and prokaryotic 
sources, has been amplified in prokaryotic 
host-vector systems. The fidelity of this 
entire process (a, b, and c) has been verified 
in several instances in which prokaryotic 
DNA segments have been cloned in E. coli 
and resulted in the synthesis of new poly- 
peptides. Thus, in such cases, the informa- 
tional content contained in the inserted pro- 
karyotic DNA is expressed as evidenced by 
the synthesis of mRNA and novel proteins. 
With few exceptions (some yeast inserts) 
the expression of eukaryotic DNA in the 
form of biologically active or biochemically 
detectable polypeptides in prokaryotes has 
not been demonstrated using chromosomal 
DNA inserts and unmodified vectors. In 
nearly all cases where the system has been 
rigorously examined, it has been shown that 
eukaryotic DNA has replicated in E. colU in 
some instances, RNA complementary to the 
inserted eukaryotic DNA has been identi- 
fied. 
Messenger RNA synthesis and function in 
E. coli. The synthesis of messenger RNA 
(mRNA) in a prokaryote, such as E. coli, 
proceeds in a linear fashion along the DNA 
template of Individual gene segments or 
groups of related genes. In nearly all cases 
examined, the mRNA molecules are the 
faithful collnear transcripts of prokaryotic 
genetic information and can be used in an 
unmodified form to direct the synthesis of 
prokaryotic polypeptides. The information- 
al content of mRNA corresponds directly to 
the nucleotide sequence of DNA in such sys- 
tems (i.e., aU nucleotides present in a pro- 
karyotic gene are transcribed into messen- 
ger RNA which, in turn, programs the syn- 
thesis of a corresponding protein). Control 
of this phase of gene expression appears to 
be solely at the level of RNA synthesis. 
In prokaryotes (and eukaryotes), nucleo- 
tide sequences preceding the sequences cor- 
responding to the actual genes play a major 
role in determining (a) whether a given 
DNA sequence will be transcribed into RNA 
and (b) whether the RNA so synthesized 
will efficiently bind to ribosomes, a prereq- 
uisite for protein synthesis. For example, 
certain DNA sequences Interact with regions 
of RNA polymerase and thereby participate 
in the initiation of RNA synthesis: they are 
not represented in the final RNA product. 
DNA sequences specifying binding to ribo- 
somes are physically located between those 
for initiation of RNA synthesis and se- 
quences encoding the amino acids of a par- 
ticular protein (the gene) and are also con- 
tained in the functional mRNA molecules. 
Messenger RNA synthesis and metabolism 
in eukaryotes. Our understanding of gene 
regulation and expression in eukaryotic 
cells has Increased markedly during the past 
10 months. A common feature of all systems 
that have been carefully evaluated is that 
the initial, faithful RNA copy of the DNA is 
extensively modified to produce a function- 
al form of mRNA. The final mRNA contains 
only a fraction of the sequences present in 
the original RNA product. That is to say. 
portions of large RNA molecules are re- 
moved by mechanisms that are, at present, 
poorly understood and the remaining seg- 
ments of the primary RNA transcript are 
then rejoined to one another. In nearly all 
cases an RNA segment containing a riboso- 
mal binding site is joined to a segment 
coding for a polypeptide; in addition, larger 
gene segments are often joined together. 
This process was first observed in animal 
virus systems 1, 2, where it was shown that 
viral mRNA, containing the information for 
a product which had been previously 
mapped to a specific locus on the viral 
genome, was complementary to regions of 
the viral DNA which were separated by 
more than a thousand nucleotides. 
Support for the concept of complex modi- 
fication leading to functional mRNA in eu- 
karyotic cells has recently come from re- 
combinant DNA experiments in which chro- 
mosomal DNA has been cloned in E. coli. 
When individual cloned eukaryotic genes 
are carefully analyzed, intervening DNA se- 
quences which Interrupt the actual - se- 
quence of the gene in chromosomal DNA 
have been identified. To date, such intra- 
genic DNA has been detected in ovalbumin 
(3, 4), 0 globin (5. 6), immunoglobulin (7), 
and even tRNA genes (8). In one instance it 
has been clearly shown that the intervening 
DNA sequences, present in the primary 
RNA transcript Of /9 globin DNA, are absent 
in 0 globin mRNA (5). These mechanisms 
presumably function in some regulatory 
fashion to modulate eukaryotic gene activi- 
ty. 
Implications tor Recombinant DNA Research 
A. The discovery of the existence of com- 
plex processes involved in the maturation of 
mRNA eukaryotic cells and the demonstra- 
tion of intragenic DNA In several eukaryotic 
genes suggests that: (1) cloning of chromo- 
somal DNA in E. coli DNA (shotgun or puri- 
fied) will poee little, if any, risk since the 
maturation mechanisms have never been 
observed in prokaryotes; and (2) investiga- 
tors who wish to develop prokaryotic clon- 
ing systems for the purpose of synthesizing 
useful biological products will utilize cDNA 
copies of functional mRNAs er synthetic 
DNA with a nucleotide sequence derived 
from a known amino acid sequence as DNA 
inserts. 
B. Vectors are currently being "engi- 
neered" to ensure efficient transcription 
and translation of DNA inserts. Using 
slightly different approaches, groups in San 
Franscisco and at Harvard (9-11) are pre- 
paring DNA segments which: (1) contain the 
sequences necessary for Interaction with E. 
coli RNA polymerase linked closely to (2) 
sequences which encode a bacterial ribo- 
some binding site. Such DNA segments can 
then be added to a prokaryotic cloning 
vector next to the site into which a foreign 
DNA will be inserted. This arrangement will 
facilitate the transcription of the Inserted 
DNA and enable the mRNA so synthesized 
to bind to bacterial ribosomes. This embel- 
lishment has already been used to maximize 
the expression of a bacteriophage gene and 
human somatostatin DNA in a plasmid 
vector system 10, 11). 
REVERENCES FOR FOOTNOTE 13 
1. Berget, S. M , Moore. C., and Sharp, P. 
A. 1977, Proc. Nat. Acad. Sci., USA 74, 
3171 3178 
2. Aloni, Y . Dhar R., Laub, O., Horowitz, 
M , and Khoury ti. 1977). Proc. Nat. Acad. 
Sci., USA 74, 3688 3690. 
3. Breathnack, R , Mandel, J. L., and 
Chambon, P. 1977,. Nature 270, 314 319. 
4. Weinstock, R., Sweet, R.. Weiss, M„ 
Cedar H , and Axel, R. (1978). Proc. Nat. 
Acad. Sci., USA 75, 1299 1301. 
5. Jeffreys, A J. and Flavell, R. A. (1977). 
CeU 12, 1097 1108. 
6. Tilghman, S. M , Tiemeier, D. C., Seid- 
man, J. O , Peterlin, B. M., Sullivan, M., 
Malzel, J. V., and Leder P. (1978). Proc. Nat. 
Acad Sci., USA 75, 725-759. 
7. Tonegawa, S.. Brack, C., Hozumi, N., 
and Schuller R. (1977). Proc. Nat. Acad. 
Sci., USA 74. 3618 3522. 
8. Valenzuela, P., Venegas, A.. Weinberg. 
F., Bishop, R , and Rutter, W. (1978). Proc. 
Nat. Acad. Sci., USA 75. 190 194. 
9. Backman, K , Ptashne, M., and Gilbert, 
W. (1976>. Proc. Nat. Acad. Sci., USA 73. 
4174 4178. 
10. Itakura, K , Hlrose, T„ Crea, R., Riggs, 
A., Heyneker H., Bolivar. F. and Boyer, H. 
(1977). Science 198, 1056-1063. 
11. Backman, K.. aqd Ptashne, M. (1978). 
CeU 13, 66-71. 
(14) Prohibition (i) in the original guide- 
lines forbids experiments with “oncogenic 
viruses classified by NCI as moderate risk." 
The absence of evidence that use of these 
viruses will lead to formation of agents 
harmful to man and the potential for ob- 
taining useful new knowledge, relevant to 
carcinogenesis in particular, and genetics in 
general, supports the removal of the prohi- 
FEDERAL REGISTER, VOL 43, NO. 145— FRIDAY, JULY 29, 1978 
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