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BIOCHEMICAL SOCIETY TRANSACTIONS
Harris, T. J. R., Lowe, P. A., Lyons, A., Thomas, P. G., Eaton,
M. A. W., Patel, T. P., Bose, C. C., Carey, N. H. & Doel,
M. T. (1982) Nucleic Acids Res. 10, 2177-2187
Moir, D., Mao, J., Schumm, J. W., Vovis, G. F., Alford, B. L. &
Taunton-Rigby A. (1982) Gene 19, 127-138.
Nishimori, K., Kawaguchi, Y., Hidaka, M., Uozumi, T. & Beppu,
T. (1982) J . Biochem. 91, 1085-1088
Rosen, J. M. (1976). Biochemistry 15, 5263-5271
Uchiyama, H., Uozumi, T., Beppu, T. & Arima, K. (1980) Agric.
Biol. Chem. 44, 1373-1381
Replacement of anticodon-loop nucleotides to produce functional tRNAs
A. G . BRUCE, J. F. ATKINS, N. WILLS, L. BARE,
0. UHLENBECK and R. F. GESTELAND
Howard Hughes Medical Institute, Department of Biology,
University of Utah. Salt Lake City, UT 84112 U.S.A..
Department of Biochemistry, University College Cork,
Ireland, and Department of Biochemistry, University of
Illinois. Urbana, IL 61801, U.S.A.
Systematic alteration of tRN A anticodon-loop nucleotides
is beginning to reveal features of codon-anticodon interaction. Two complementary approaches are being used.
Alterations at the D N A level provide positional flexibility,
but in some situations may be constrained by a processing
requirement. Alterations at the t R N A level have only been
demonstrated at more restricted sites, but at these sites are
only constrained by the need for amino-acylation. Yarus
and co-workers have recently performed elegant studies on
the modification of the anticodon loop region of the
Escherichia coli tRNATrps u + 7gene (Thompson et al., 1982).
Our approach has been to apply the ‘recombinant RNA’
procedures described by Bruce & Uhlenbeck (1982). Other
workers have developed similar protocols for a number of
different yeast and E. coli tRNAs for the study of aminoacylation and tRNA modification (Carbon et a[., 1982;
Schulman et al., 1983).
Our initial experiment was to make derivatives of yeast
tRNAPhewith a C U A anticodon, complementary to the
amber (UAG) terminator. Functional amber suppressor
tRNAs were produced and their activity assayed (Gesteland et al., 1976) in a mammalian cell-free-proteinsynthesizing system. tRNAs were constructed with A, C, U
or G on the 3’ side of the C U A mticodon. The tRNA
containing the purines were efficient amber suppressors,
whereas those containing pyrimidines were inefficient
(Bruce et al., 1982). This is consistent with the fact that a
modified purine is always found at position 37, and
strengthens the view that the nucleotide at position 37
stabilizes the codon-anticodon interaction by increasing the
stacking.
A second series of experiments involving alterations of
the position 33 nucleotide in yeast tRNATyr gave more
surprising results (Bare et al., 1983). Uridine at position 33 is
one of the most conserved nucleotides in tRNA. Cytidine
can effectively substitute for uridine and even tRNAs with a
purine at position 33 are not more than 3-fold less effective
as suppressors. The constant uridine at position 33 is not
obligatory, but if it allowed a small increase in translation
efficiency it would not be detected in our assay. Nevertheless such an increase could be adequate to maintain a strong
selection.
Previously it has been shown that an excess of certain
normal tRNAs in E. coli extracts greatly enhances ribosomal frameshifting during translation of natural m R N As.
The effect of each of these tRNAs is completed by a specific
different tRNA (Atkins et al., 1979). We plan to try to
extend the procedure of anticodon-loop replacement to at
least one of the ‘shifty’ tRNAs to help distinguish between
different models of the codon-anticodon interaction.
Atkins, J. F., Gesteland, R. F., Reid, B. R. & Anderson, C. W.
(1979) Cell 18, 1119-1131
Bare, L., Bruce, A. G., Gesteland, R. F. & Uhlenbeck, 0.C. (1983)
Nature (London) 305, 554556.
Bruce, A. G. & Uhlenbeck, 0. C. (1982) Biochemistry 21, 855-861
Bruce, A. G., Atkins, J. F., Wills, N., Uhlenbeck, 0. C. &
Gesteland, R. F. (1982) Proc. Natl. Acad. Sci. U . S . A .79, 71277131
Carbon, P., Haumont, E., De Henau, S., Keith, G. & Grosjean, H.
(1982) Nucleic Acids Res. 10, 3715-3732
Gesteland, R. F., Wolfner, M., Grisafi, P., Fink, G., Botstein, D.
& Roth, J. R. (1976) Cell 7, 381-390
Schulman, L. H., Pelka, H. & Susani, M. (1983) Nucleic Acids. Res.
11, 1439-1446
Thompson, R. C., Cline, S. W. & Yarus, M. (1982) in Interaction of
Transcriptional and Translational Controls in the Regulation of
Gene Expression (Grunberg-Manago, M. & Safer, B., eds.), pp.
189-202, Elsevier Science, New York
Molecular studies on the DNA-repair genes of the cyanobacterium Gloeocupsu dpicolu
D. NUTTALL, C. GEOGHEGAN and
J. A. HOUGHTON
Department of Microbiology, University College, Galway,
Ireland
It is well established that cells have the capacity to recover
from radiation-induced damage. In bacteria, three processes of D N A repair are found : photoreactivation,
excision repair and post-replication repair. Photoreactivation involves the enzymatic splitting of cyclobutyl pyrimidine dimers in the presence of near-U.V. or visible light.
One type of excision repair removes the modified base by an
N-glycosidase and the chain is then nicked by an apurinic
(apyrimidinic) acid endonuclease. In Escherichia coli the
excision-repair process is catalysed by the combined action
of the products of uvrA, uvrB and uvrCgenes and seems to be
part of the u.v.-inducible SOS response, which itself
depends on the recA + and lexA+ functions. Post-replication
repair is also recA+-dependent in E. coli.
Previous studies have indicated the presence of photoreactivation, excision repair and an inducible response to
U.V. radiation in the unicellular cyanobacterium Gloeocapsa alpicola. As a result of these physiological studies, it
was decided to investigate the repair processes at the
molecular level. The initial approach was to determine if
there was sufficient homology between the genomic D N A
of G. alpicola and the excision-repair genes of E. coli to
permit heterologous hybridization. Genomic D N A was
digested with restriction enzymes and transferred to nitrocellulose filters by Southern blotting, the blots were then
hybridized with 32P-labelledprobes carrying the uvrA, uvrC
and recA genes of E. coli respectively. These preliminary
1984