- Email: [email protected]
Modulation of a Drosophila melanogaster tRNA gene transcription in vitro by a sequence TNNCT in its 5′ flank
Gene, 60 (1987) 13-19
13
Elsevier GEN 02180
Modulation of a Drosophila in its 5’ flank (Recombinant
DNA;
mefunogaster tRNA gene transcription in vitro by a sequence TNNCT
site-specific
mutagenesis;
gene expression;
tRNAy”‘;
RNA polymerase
III)
Fereydoun G. Sajjadi b* and George B. Spiegelman” ” Departments
of Medical
B. C. (Canada
Genetics and Microbiology,
and ’ Program
in Genetics,
University of British Columbia,
Vancouver,
V6T 1 W5)
Received
27 May 1987
Accepted
31 July 1987
SUMMARY
We have previously demonstrated the existence of both positive and negative transcription modulatory sequences in the 5’ flank of a Drosophila melanogaster tRNAy”’ gene using deletion analysis. The deletion of a pentadeoxynucleotide TCGCT between nucleotides (nt) -34 and -38 relative to the mature coding sequences resulted in a 90% decrease in template activity. In the present study, we have created a number of site-specific changes in the sequence TCGCT. Results indicate a significant decrease in the level of template activity when nt -38(T), -35(C) and -34(T) were mutated. In contrast, a change at nt -37(C) resulted in a slight increase in transcription and a change at nt -36(G) reduced template activity by only 1%. Sequences flanking other tRNA genes which are efficient templates for transcription were examined and found to contain a sequence closely related to TCGCT. We propose that a general form of the sequence TNNCT is a positive transcription modulator for a class of Drosophila tRNA genes.
INTRODUCTION
Transcription efficiency of tRNA genes has been shown to be dependent upon sequences contained in their 5’-flanking regions in addition to the conserved
Correspondence to: Dr. G.B. Spiegelman, biology,
University
(Canada
V6T 1W5) Tel. (604)228-2036.
* Present
address:
California
San
of
British
Department Diego,
La
Department
Columbia,
of Micro-
Vancouver,
B.C.
of Biology, B-022, University Jolla,
CA
92093
of
(U.S.A.)
Tel. (619)534-2327. Abbreviations:
bp, base pair(s); nt, nucleotide(s);
RNA; J’,,,,,, calculated tRNA gene template.
0378-I 119/87/$03.50
0
maximum
tRNA, transfer
transcription
1987 Elsevier Science Publishers
rate
from
a
B.V. (Biomedical
internal promoter sequences (Ciliberto et al., 1982; Sprague et al., 1980; Rajput et al., 1982; Larson et al., 1983; Raymond and Johnson, 1983; Shaw and Olson, 1984; Schaack et al., 1984; Sajjadi et al., 1987). For example, sequences located between nt -34 and -11 of a Bombyx mori tRNAA’” gene (Larson et al., 1983) and nt -22 to -2 of a yeast tRNAp” gene (Raymond and Johnson, 1983) are required for efficient transcription in vitro. In addition, up to 36 nt of wild-type flanking sequence are required for efficient template activity for a yeast tRNATy’ gene in vivo (Shaw and Olson, 1984). The most extensive studies on the effects of sequences contained in the 5’-flank on transcription have been carried out with two different Drosophila tRNA genes Division)
14
(Schaack the
et al., 1984; Sajjadi et al., 1987). However,
mechanism
modulate
tRNA
Our previous
by which
5’-flanking
transcription
remains
studies
M13K07
mature coding sequence
resulted in a large decrease
in the level oftranscription
of these genes. Disruption
between
nt -34
and TCGCT
and -38
major effect in reduction
(pV4a.5179)
was responsible of template
for the
activity.
Fur-
thermore, the comparison of their 5’-flanking sequences revealed no obvious additional homology. These observations suggested that a pentadeoxynucleotide of the sequence TNNCT where N is any nt, may be a positive modulator of transcription of these genes. To elucidate the role of this sequence in tRNA transcription, we have created a number of site-specific nt changes in the TCGCT sequence in pV4a.5-138, which contains the tRNAy”’ gene and 138 nt of 5’-flanking region. Our results confirm that the sequence TNNCT is an important positive modulator of tRNA transcription. In addition, the 5 ‘-flanking sequences of other tRNA genes were searched for the presence of a sequence of the form TNNCT. The presence of the sequence between nt -25 and -40 was found to be strongly correlated with efficient in vitro transcription for Drosophila tRNA genes.
MATERIALS
AND
(1985).
pared
et al. (1984) using pArg which
(pArg)
dU-containing
Kunkel
unknown.
contains a tRNAArg gene had shown that deletion of the 5’-flanking regions up to nt -32 relative to the
of the TTTCT
using
179 (Sajjadi
a Drosophila tRNA,V”’ gene
et al., 1987) containing and those of Shaack
with pV4a.5
sequences
METHODS
according
templates
Single-stranded to Dente
(Pharmacia)
as described template
by
was pre-
et al. (1983) except that
was used in place of IRl
helper phage. Forward and reverse sequencing primers were obtained from Pharmacia. The following oligodeoxynucleotides, synthesized on
an Applied
Synthesizer Biochemistry,
Biosystems
Model
380-A
by T. Atkinson (Department University of British Columbia),
used to obtain
specific nucleotide
changes
DNA of were
between
nt -34 and -38: 5’-CAGTTGAGGGCGCTGAAC-3’; 5’-GTTGAGGTCGATGAAGTTGGC-3’; 5’-GTTGAGGTCGCAGAAGTTGGCC-3’; 5’-GTTGAGGGCGATGAAGTTG-3’; 5’-GTTGAGGGCGGTGAAGTTG-3’; 5’-GCAGTTGAGGTAGCTGAAGTTG-3’; 5’-GCAGTTGAGGTCTCTGAAGTTG-3’. During the mutagenesis experiment it became clear that mutagenic oligodeoxynucleotides were annealing to a secondary sequence in the bacteriophage fl origin of replication and thereby severely decreasing the number of transformants obtained. This was overcome by subcloning the EcoRI-Hind111 fragment into pTZ 19U (U.S. Biochemical) and extending primer synthesis away from the secondary hybridization site. Mutants were identified by dideoxy sequence analysis (Sanger et al., 1977). Rates of mutagenesis varied between 30 and 75 % of recovered clones. Mutants were subsequently recloned into pEMBL8- for transcription analysis. DNA used in transcription assays was purified by banding in CsCl and judged to be at least 75% supercoiled in agarose gels.
(a) Plasmid constructs
(c) In vitro transcription assays
The construction of pV4a.5 plasmids has been previously described (Sajjadi et al., 1987). The tRNA,V”’ gene containing 138 bp of 5’-flank was liberated by cleavage with EcoRI and HindHI. This 286-bp fragment was subcloned into pEMBL8(Dente et al., 1983) to create pV4a.5-138.
In vitro transcription assays were carried out as previously described (Sajjadi et al., 1987; St. Louis and Spiegelman, 1985). Each series of transcriptions contained ten input points ranging from 10 to 100 ng of template DNA and nonspecific DNA to a final concentration of 1 pg/50 ~1. Each mutant template was transcribed alongside a pV4a.5- 138 series which served as a control. The maximum level of transcription directed by each template (V,,,) was calculated by both a numerical and a graphical procedure (St. Louis and Spiegelman, 1985) and the
(b) In vitro mutagenesis Site-specific double-primer
mutagenesis was carried out by the method of Zdller and Smith (1984)
15
change in V,,, reported as a y0 increase or decrease over the transcription of the unmutated wild-type
phoretic
template
pV4a.5-138
carried
were repeated
out in parallel.
with two different
the Y0 changes in V,,, between the extracts.
All transcriptions S- 100 extracts
reported
and
as the average
Fig. 1A shows separation and
observed.
(a) A pentadeoxynucleotide of tRNAy
in the 5’4lanking region
gene determines the efficiency of tran-
scription The subclone of pV4a.5179 (Sajjadi et al., 1987), pV4a.5-138 was chosen for site-directed mutagenesis of the sequence TCGCT contained between nt -34 and -38 relative to the mature coding sequence. The mutants created and their transcription efficiencies relative to pV4a.5138 are listed in Table I. All mutant tamplates were transcribed in parallel with a set of pV4a.5138 DNA controls. Reactions contained between 10 and 100 ng of template DNA; the very low template input is an important technical consideration as previously shown by Wilson et al. (1985) and St. Louis and Spiegelman (1985). The Vmax values were derived from the intercept of a least-squares line of the cpm of transcript plotted vs. DNA input, as previously described (St. Louis and Spiegelman,
TABLE Change
1985).
of transcription the double
This product
products
mutation
from
nt -38(G);
had previously
been shown
at a G at nt -9 (Sajjadi
et al., 1987).
Fig. 1B shows a plot of the reciprocal
of the cpm of
transcript
AND DISCUSSION
of the electro-
-35(A). As shown in this figure, and in all other instances, only a single transcription product was to initiate
RESULTS
autoradiograms
vs. the reciprocal
of the DNA input which
allowed the derivation of V,,, values from the intercepts of the least-squares line describing the data. The data in Fig. 1B denlpnstrate
that the nucleotide
substitutions in the 5’-flanking DNA of pV4a.5-138 reduce the template efficiency significantly. Table I summarizes the average of the y0 change in V,,, values for each mutated template. Templates containing single nucleotide changes in the TCGCT sequence, i.e. at nt -34(T), -35(C) and -38(T) each resulted in approx. 30% decrease in V,,,. A double change at nt -35(C) and -38(T) resulted in a V,,, approx. 40% lower than wild type. In contrast, the single base substitution at nt -37(C) resulted in a small increase in V,,, relative to wild-type sequence. Furthermore, a change at nt -36(G) decreased transcription by only 1%. Together these latter two changes suggest that nt -37(C) and -36(G) are not important for transcription modulation (Sajjadi et al., 1987). The increase in V,,, seen with the change at nt -37(C) may be due to the increased A + T content of the 5’-flank. In contrast, the trans-
I in V,,,
Template
values obtained
from in vitro transcription
of TNNCT
mutants
a
relative
to pV4a.5-138
v max b y0 change
pV4a.5-138
TCGCT
(wild type)
GCGCT
[mutation,
nt -38(G)]
128.2
TCGAT
[mutation,
nt -35(A)]
TCGCA
[mutation,
nt -34(A)]
132 J32.8
GCGGT
[mutation,
nt -38(G);
-35(C)]
137.7
GCGAT
[mutation,
nt -38(G);
-35(A)]
TCTCT
[mutation,
nt -36(T)]
142 1 1.1*
TAGCT
[mutation,
nt -37(A)]
t12.6
a These point mutants
0 (control)
are described
in RESULTS
AND
DISCUSSION,
section a.
experiments for each of the mutants and is lJ VW was calculated from the data obtained from ten-point DNA input transcription expressed as y0 increase (upward arrows) or decrease (downward arrows) over the value or pV4a.5-138 transcribed in parallel. V,,,,, values are the average S-100 extract.
of determinations
using two different
S-100 extracts.
If,,,,, value with an asterisk
was calculated
using a single
16
A wild-type 2
1
3
4
5
6
-38G;-35A 7
8
9
10
Fig. 1. In vitro transcription pV4a.5-138
products,
[-38(G);
(B) Double reciprocal of product oftemplate
of pV4a.5138
and of the double mutant
respectively.
plot of transcription by Cerenkov
Transcriptions
-35A
products transcription
The data were plotted
DNA). The lines were derived by the method ofleast
and 0.999 for -38G,
at nt -38(G);
4
5
6
squares
-35(A).
(A) Autoradiogram
8
9
10
100
of a polyacrylamide
gel
of IO to 100 ng of pV4a.5-138
and
in MATERIALS
products
AND METHODS,
section c.
were excised (see panel A) and the amount
as l/V (V = cpm of transcript/h with correlation
7
75
from transcription
were at 24°C as described
data. Gel slices containing counting.
3
50
Lanes I-10 and 1 I-20 show the radiolabeled
-35(A)],
was determined
2
25
-25
of transcription
1
coefficients
of reaction)
vs. l/S (S = mass
of 0.998 for pV4a.5138
(triangles),
(squares).
version of a C to an A at nt -35 resulted in a 32% decrease in V,,,, emphasizing the specificity of the TCGCT sequence at nt -35(C). In our earlier paper (Sajjadi et al., 1987), we observed a more dramatic decrease in the level of transcription when the nucleotides 5’ to and ineluding TCGCT were removed and replaced by
vector sequences when compared to site-specific changes in TCGCT in Table I. The results presented here implicate the short nucleotide stretch immediately 5’ to TCGCT (to nt -45) as a possible secondary positive modulator of transcription as we previously suggested.
17
(b) Comparison
sequences of Droso-
of 5’-flanking
drop in transcription.
Together,
these data led us to
phila tRNA genes and their respective transcription
believe that a sequence with a general form TNNCT
efficiencies
would be a candidate
for further
examination.
We
therefore
searched the 5’-flanking region of other Drosophila tRNA genes for the presence of TNNCT
Data presented in Table I illustrate that nt -38(T), -35(C) and -34(T) are essential for maximal stimulation
of transcription
transversions nificantly
of pV4a.5-138,
at nt -37(C)
decrease
do not sig-
the rate of transcription.
on pArg (Schaack
and
TTTCT
TNNCT
between
transcribed.
Studies
S011, 1985) indicated
deletion of the sequence
TABLE
and -36(G)
sequences (Table II). Valine and arginine
whereas
tRNA
nt -30
In a previous
genes which have a
and -40
are efficiently
study, we discovered
that
a deletion endpoint pV4a.5-20R which acquired a TNNCT by a combination of vector and 5’-flanking
that
resulted in an 88 %
II
Correlation
of transcription
Template
il
efficiency Position
and the pentadeoxynucleotide
of TNNCT’
pYH48-Arg
- 38TTTCT
pEl.R-Arg
- 52TTGCT
- 40TATCT
d
- 88TCTCT
- 84TTTCT
- 69TTGCT
- 36TGCCT
d
pDt67R-Arg
- 15 ITAACT
pDt l7R-Arg
- 89TGACT
- 126TAGCT
in the 5’-flanking
region of a tRNA
Transcription’
Reference
+
Schaack
gene
et al. (1984)
+e +e
- 106TGTCT
- SOTTCCT - 53TCCCT
- 38TTGCT
- 33TTTCTd
p 11 F-Arg
No TNNCT
(to -40)
p35D-Arg
No TNNCT
pE3.8-Arg
No TNNCT
pl7D-Arg
No TNNCT
pAva4-Arg
No TNNCT“
-(+I -(+I
-(+P _
Dingermann
et al. (1982)
Dingermann
et al. (1982)
Dingermann
et al. (1982)
_e
pAsn8-Asn
- 42TGGCT
- 37TGGCT
pAsn6-Asn
No TNNCT
(to -48)
pAsnl-Asn
No TNNCT
(to -48)
pHis-His
- 46TTGCT
p48His- YHis
- 22TTGCT
pDt39R-:-Y”
- 36TACCT
pDt59R-;-Y” pDt5-Scr pDtlhH#
TNNCT
+
Lofquist
and Sharp
(1986)
and Sharp
(1986)
Lofquist
and Sharp
(1986)
- 38TTCGT
--(+I _ + -(+) + +
Lofquist
No TNNCT
+
St. Louis and Spiegelman
(1985)
+(-I +(-I + +
St. Louis and Spiegelman
(1985)
St. Louis and Spiegelman
(1985)
I-;;;
~ 39TAGCT
pDt 16H # 5-T;;
No TNNCT
pDt55-0.6-,V”’
- 39TGGCT
pV4a.5-179-T”’
- 38TCGCT
- 23TCTCT
pDt92R-,V”’
- 79TTACT
- 48TTGCT
Cooley et al. (1984) Cooley et al. (1984)
+(-I”
- 44TAACT
DeFranco
et al. (1982)
DeFranco
et al. (1982)
Rajput
et al. (1982)
Sajjadi
et al. (1987)
Addison
et al. (1982)
- 39TATCT pDt78R-:;’
No TNNCT
“ Designation
of recombinant
plasmids
or absence
of the positive
’ The presence Numbers
indicate
’ Symbols
the position
for transcription
- ( + ), inefficient is taken
c L. Duncan
containing
J. Leung,
and G.B.S.,
Drosophila tRNA
modulatory
of the 5’ T in the TNNCT effciences
-
TNNCT
sequence
in the 5’-flanking
, no detectable
and G.M. Tener, personal results.
transcription.
communication.
species is indicated
in the 5’-flanking
10% of pV4a.5-179);
the levels do not relate to pV4a.5-179. unpublished
genes. lsoacceptor
sequence
are: + , efficient (within
(less than 1 y0 of pV4a.5.179);
from the literature,
d C.H. Newton,
Leung et al. (1984) after the hyphen.
region of various
DNA, relative to the mature + (- ), inefficient
tRNA genes is shown. coding sequence
( + 1).
(less than 15% of pV4a.5-179);
For entries where evaluation
of transcription
efficiency
IS
sequences
at position
-20 relative to the gene was an
inefficient template when compared to pV4a.5179 (Sajjadi et al., 1987). This would seem to be similar to the pseudohistidine TNNCT
tRNA
gene
which
has
located at nt - 18 to -22 and transcribed
a at
other correspondences random frequencies.
which occurred
We would emphasize, TNNCT tRNA
that we do not believe that
is the only positive transcription genes.
As
at the above
discussed
in
effector for
RESULTS
AND
tRNAHi” gene on plasmid pHis (Cooley et al., 1984).
DIscussIoN, section a, our previous analysis suggested that nt -46 to -42 in pV4a.5-179 [AGTTG]
Plasmid
may
less than
1% of the efficiency pHis has a TNNCT
at nt -42 and -46 and
is an effective in vitro template. that of the tRNA,V”’ allogene This plasmid
is a moderately
of its counterpart
An unusua1 case is on plasmid
good template
and carries five copies of the TNNCT. tRNAA’g
gene (pDt17R)
was found
pDt92R. in vitro
Recently,
a
to transcribe
very efficiently with eight copies of TNNCT in its 5’-flank (C.H. Newton, J. Leung and G.M. Tener, personal communication). Genes coding for tRNAVa’ and tRNAArb which do not contain the TNNCT in their 5’-flank either fail to direct transcription or direct transcription very inefficiently in vitro. The TNNCT is also found in the 5’-flanking regions of other tRNA genes such as tRNAF (DeFranco et al., 1982) which directs efficient transcription and tRNAi’” and tRNALe” (Robinson and Davidson, 198 1) whose transcription has not been studied. It should be noted that two TNNCTs, as are found in the 5’-flank ofpV4a.5179, are not necessary to stimulate transcription, since phrg and other tRNA genes function as in vitro templates with only one copy. An important question raised by Table II is whether the occurrence of TNNCT is random in the 5’-flanking sequences of tRNA genes. The sequence does appear with expected random frequency (l/64) in the plasmid pBR322 (not shown). In a block of nt -25 to -45 relative to a tRNA gene, a single TNNCT sequence should occur with a Poisson frequency of 0.22. Of the 23 genes in Table II, 13/23 ( = 0.56) have a TNNCT in the nt -25 to -45 region while ten do not. Furthermore, when analysed as groups, of the genes which are transcribed at moderate to high efficiency, twelve of the 14 have TNNCT sequences in the nt -25 to -45 region. Conversely, of the genes which are transcribed at less than 1% of pV4a.5-179, none have a TNNCT sequence in the nt -25 to -45 region. Thus, the association of the sequence TNNCT with genes which are active templates is not random using our current data. We examined the 50 nt 5’ to the genes in Table II for other homologies and could find no
also
stimulate
transcription
1987). We have preliminary analysis
that
scription
of the tRNAs”
the same
G.B.S., unpublished
(Sajjadi
et al.,
evidence from a deletion sequence
stimulates
gene in pDt5 (F.G.S.
observations).
expect all genes with a TNNCT
tranand
Thus, we do not in their 5’-flanking
sequence to be transcribed equally well and there are genes without the TNNCT sequence which are effective in vitro templates. For example, pDt5 (Table II; St. Louis and Spiegelman, 1985), directs transcription equivalent to pV4a.5-179 in the absence of TNNCT. On the other hand, for one tRNASe’ allogene (pDt 16H No. 1; St. Louis and Spiegelman, 1985), a deletion mutation which removed .5’-Banking sequences distal to nt -36 disrupted a TNNCT and resulted in a four-fold decrease in V,,,. In addition, it may be that pDt5 would serve as a more efficient template if a TNNCT were placed between nt -25 and -45 in its 5’-flanking region. It does appear that multiple TNNCT sequences do not provide additional stimulation of transcription since pDt92R-Val, is not a better template than pV4a.5179, but has more copies of the TNNCT sequence. Experiments investigating the mechanism of the effect of TNNCT on transcription are in progress. Finally, other examples of 5’-flanking sequences which affect transcription of class-III genes have been found in other lower eukaryotes (Larson et al., 1983; Raymond and Johnson, 1983; Shaw and Olson, 1984). These sequences have no homology to TNNCT. Similar kinds of sequences have yet to be identified in higher eukaryotes although given the complexity of the class-III gene family and of the transcription apparatus for these genes, we would expect such sequences to exist.
ACKNOWLEDGEMENTS
We would like to thank Loverne Duncan for the preparation of S-100 extracts, David Goodin for the
19
gift of Escherichia coli BW313 and helpful suggestions on site-directed mutagenesis. This work was supported by Medical Research Council of Canada Grant 68-7364.
Rajput,
B., Duncan,
L., DeMille,
D., Miller
Jr.,
R.C.
and
Spiegelman, G.B.: Transcription of cloned transfer RNA genes from Drosophila melanogaster in a homologous cell-free extract.
Nucl. Acids Res. 10 (1982) 6541-6550.
Raymond,
G.R. and Johnson,
sequences
J.D.: The role of non-coding
in transcription
and processing
DNA
of a yeast tRNA.
Nucl. Acids Res. 11 (1983) 5969-5988. Robinson,
R.R. and
sequences. Addison,
W.R.,
Hayashi,
Astell,
C.R.,
Delaney,
A.D.,
S., Miller Jr., R.C., Rajput,
Gillam,
I.C.,
B., Smith, M., Taylor,
D.M. and Tener, G.M.: The structures
G., Castagnoli,
L., Melton,
of a eukaryotic
noncontiguous
D.A.
tRNAPro
Cortese,
R.:
gene is composed
and
of
regions. Proc. Natl. Acad. Sci. USA 79 (1982)
Cooley, L., Schaack,
J., Johnson-Burke,
D.: Transcription pseudogene. DeFranco, R.C.
factor binding
D., Thomas, gene
and
a
Siill, D.: Genes
tRNA”‘$
S., Tener, G.M., Miller Jr., Drosophila
melunogaster. Nucl. Acids Res. 10 (1982) 5799-5808. Dente,
L., Cesarini,
of single
G. and Cortese,
R.: pEMBL:
plasmids.
Acids
stranded
Nucl.
Res.
T., Johnson-Burke,
genes control extract. Kunkel,
D., Sharp,
11 (1983)
S., Schaack,
J. and
of Drosophila tRNAArs
sequences
their in vitro transcription
in Drosophila cell
without
Rapid
and
phenotype
efficient
selection.
site-specific
Proc. Natl. Acad. Sci. USA 82
A short 5’-flanking Acad.
J., Young, L.S. and Sprague,
region containing
for silkworm
conserved
K.U.:
sequences
is
alanine tRNA gene activity. Proc. Nat].
W.R., Delaney,
Jr., R.C., Spiegelman,
A.D., MacKay,
G.B., Grigliatti,
R.M., Miller
T.A. and Tener, G.M.:
Drosophila melanoguster tRNAY,“’ genes and their allogenes. A. and
Drosophila
Sharp,
melanogaster
RNA polymerase
S.: The tRNA:“”
5’-flanking
sequences
genes differentially
III. J. Biol. Chem. 261(1986)
with
S., Dingermann,
T., Johnson-Burke,
5’- and
3’-flanking
sequence
and stable complex
dependence
formation.
D., tRNA
for tran-
J. Biol. Chem. 259
(1984) 1461-1467. of a Drosophila tRNAArs
J. and Sol], D.: Transcription
gene in yeast extract: transcription Sharp,
5’-flanking
sequence
dependence
for
system. Nucl. Acids Res. 13
in a heterologous
S., Dingermann,
T., Schaack,
D.: Transcription
J., DeFranco,
of eukaryotic
tRNA
D. and Still,
genes
of control regions using a competition
in vitro,
I.
assay. J. Biol.
Chem. 258 (1983) 2440-2446. Shaw,
K.J. and
sequences
Olson,
M.V.:
Effects
of altered
5’-flanking
of a Saccharomyces
on the in vivo expression
K.U., Larson,
signals required
of
arrest
14600-14606.
D. and Morton,
D.: 5’-flanking
for activity of silk-worm in vitro transcription
sequence
alanine tRNA genes
systems.
Cell 22 (1980)
171-178. St. Louis, D. and Spiegelman, of cloned tRNAs”’
region
G.B.: Steady-state
kinetic analysis
genes from Drosophila melanogasier. Eur.
148 (1985) 305-313.
Wilson, E.T., Larson, controls
D., Young, L.S. and Sprague, tRNA
gene transcription.
K.U.: A large
J. Mol. Biol. 183
(1985) 153-163. Ziiller,
Gene 34 (1984) 207-217. Lofquist,
gene.
J. Biochem.
Sci. USA 80 (1983) 3416-3420.
Leung, J., Addison,
A.R.: DNA seqencing
Proc. Nat]. Acad. Sci. USA 74
L. and Siill, D.: The extent of a eukaryotic
in homologous
D., Bradford-Wilcox,
required
inhibitors.
J., Sharp,
Sprague,
mutagenesis
(1985) 488-492. Larson,
S. and Coulson,
crrevisiae tRNATy’ gene. Mol. Cell. Biol. 4 (1984) 657-665.
J. Biol. Chem. 257 (1982) 14 738-14 744. T.A.:
its transcription
206 (1987) 279-284.
(1977) 5463-5467. Schaack,
Analysis
Siill, D.: The 5’-flanking
G.B.: Identiti-
region of a Drosophila
in the 5’-flanking
(1985) 2803-2814.
a new family
1645-1655. Dingermann,
F., Nicklen,
chain-terminating
Schaack,
for tRNAkYS from
Miller Jr., R.C. and Spiegelman,
in vitro. Mol. Gen. Genet.
scription
Mol. Cell. Bio. 4 (1984) 2714-2722.
D., Burke, K.B., Hayashi, and
B. and Soll,
is limited by the 5’-flanking
of Drosophila tRNA”‘”
regions
F.G.,
Cooley,
1195-1199.
intervening
Cell 23 (198 1) 25 l-259.
cation of sequences
Sanger,
251 (1982) 670-673. Promoter
Sajjadi,
of a Drosophila
D.: Analysis
two tRNALe” genes contain
melanogaster tRNA,V”’ gene that modulate
of genes hybridizing
from Drosophila melanoguster. J. Biol. Chem.
with tRNAy”’ Ciliberto,
Davidson,
tRNA gene cluster:
REFERENCES
M.J. and
genesis: and
Smith,
M.: Oligonucleotide
a simple method
a single-stranded
479-488. Communicated
by S.T. Case.
directed
using two oligonucleotide DNA
template.
DNA
mutaprimers
3 (1984)
13
Elsevier GEN 02180
Modulation of a Drosophila in its 5’ flank (Recombinant
DNA;
mefunogaster tRNA gene transcription in vitro by a sequence TNNCT
site-specific
mutagenesis;
gene expression;
tRNAy”‘;
RNA polymerase
III)
Fereydoun G. Sajjadi b* and George B. Spiegelman” ” Departments
of Medical
B. C. (Canada
Genetics and Microbiology,
and ’ Program
in Genetics,
University of British Columbia,
Vancouver,
V6T 1 W5)
Received
27 May 1987
Accepted
31 July 1987
SUMMARY
We have previously demonstrated the existence of both positive and negative transcription modulatory sequences in the 5’ flank of a Drosophila melanogaster tRNAy”’ gene using deletion analysis. The deletion of a pentadeoxynucleotide TCGCT between nucleotides (nt) -34 and -38 relative to the mature coding sequences resulted in a 90% decrease in template activity. In the present study, we have created a number of site-specific changes in the sequence TCGCT. Results indicate a significant decrease in the level of template activity when nt -38(T), -35(C) and -34(T) were mutated. In contrast, a change at nt -37(C) resulted in a slight increase in transcription and a change at nt -36(G) reduced template activity by only 1%. Sequences flanking other tRNA genes which are efficient templates for transcription were examined and found to contain a sequence closely related to TCGCT. We propose that a general form of the sequence TNNCT is a positive transcription modulator for a class of Drosophila tRNA genes.
INTRODUCTION
Transcription efficiency of tRNA genes has been shown to be dependent upon sequences contained in their 5’-flanking regions in addition to the conserved
Correspondence to: Dr. G.B. Spiegelman, biology,
University
(Canada
V6T 1W5) Tel. (604)228-2036.
* Present
address:
California
San
of
British
Department Diego,
La
Department
Columbia,
of Micro-
Vancouver,
B.C.
of Biology, B-022, University Jolla,
CA
92093
of
(U.S.A.)
Tel. (619)534-2327. Abbreviations:
bp, base pair(s); nt, nucleotide(s);
RNA; J’,,,,,, calculated tRNA gene template.
0378-I 119/87/$03.50
0
maximum
tRNA, transfer
transcription
1987 Elsevier Science Publishers
rate
from
a
B.V. (Biomedical
internal promoter sequences (Ciliberto et al., 1982; Sprague et al., 1980; Rajput et al., 1982; Larson et al., 1983; Raymond and Johnson, 1983; Shaw and Olson, 1984; Schaack et al., 1984; Sajjadi et al., 1987). For example, sequences located between nt -34 and -11 of a Bombyx mori tRNAA’” gene (Larson et al., 1983) and nt -22 to -2 of a yeast tRNAp” gene (Raymond and Johnson, 1983) are required for efficient transcription in vitro. In addition, up to 36 nt of wild-type flanking sequence are required for efficient template activity for a yeast tRNATy’ gene in vivo (Shaw and Olson, 1984). The most extensive studies on the effects of sequences contained in the 5’-flank on transcription have been carried out with two different Drosophila tRNA genes Division)
14
(Schaack the
et al., 1984; Sajjadi et al., 1987). However,
mechanism
modulate
tRNA
Our previous
by which
5’-flanking
transcription
remains
studies
M13K07
mature coding sequence
resulted in a large decrease
in the level oftranscription
of these genes. Disruption
between
nt -34
and TCGCT
and -38
major effect in reduction
(pV4a.5179)
was responsible of template
for the
activity.
Fur-
thermore, the comparison of their 5’-flanking sequences revealed no obvious additional homology. These observations suggested that a pentadeoxynucleotide of the sequence TNNCT where N is any nt, may be a positive modulator of transcription of these genes. To elucidate the role of this sequence in tRNA transcription, we have created a number of site-specific nt changes in the TCGCT sequence in pV4a.5-138, which contains the tRNAy”’ gene and 138 nt of 5’-flanking region. Our results confirm that the sequence TNNCT is an important positive modulator of tRNA transcription. In addition, the 5 ‘-flanking sequences of other tRNA genes were searched for the presence of a sequence of the form TNNCT. The presence of the sequence between nt -25 and -40 was found to be strongly correlated with efficient in vitro transcription for Drosophila tRNA genes.
MATERIALS
AND
(1985).
pared
et al. (1984) using pArg which
(pArg)
dU-containing
Kunkel
unknown.
contains a tRNAArg gene had shown that deletion of the 5’-flanking regions up to nt -32 relative to the
of the TTTCT
using
179 (Sajjadi
a Drosophila tRNA,V”’ gene
et al., 1987) containing and those of Shaack
with pV4a.5
sequences
METHODS
according
templates
Single-stranded to Dente
(Pharmacia)
as described template
by
was pre-
et al. (1983) except that
was used in place of IRl
helper phage. Forward and reverse sequencing primers were obtained from Pharmacia. The following oligodeoxynucleotides, synthesized on
an Applied
Synthesizer Biochemistry,
Biosystems
Model
380-A
by T. Atkinson (Department University of British Columbia),
used to obtain
specific nucleotide
changes
DNA of were
between
nt -34 and -38: 5’-CAGTTGAGGGCGCTGAAC-3’; 5’-GTTGAGGTCGATGAAGTTGGC-3’; 5’-GTTGAGGTCGCAGAAGTTGGCC-3’; 5’-GTTGAGGGCGATGAAGTTG-3’; 5’-GTTGAGGGCGGTGAAGTTG-3’; 5’-GCAGTTGAGGTAGCTGAAGTTG-3’; 5’-GCAGTTGAGGTCTCTGAAGTTG-3’. During the mutagenesis experiment it became clear that mutagenic oligodeoxynucleotides were annealing to a secondary sequence in the bacteriophage fl origin of replication and thereby severely decreasing the number of transformants obtained. This was overcome by subcloning the EcoRI-Hind111 fragment into pTZ 19U (U.S. Biochemical) and extending primer synthesis away from the secondary hybridization site. Mutants were identified by dideoxy sequence analysis (Sanger et al., 1977). Rates of mutagenesis varied between 30 and 75 % of recovered clones. Mutants were subsequently recloned into pEMBL8- for transcription analysis. DNA used in transcription assays was purified by banding in CsCl and judged to be at least 75% supercoiled in agarose gels.
(a) Plasmid constructs
(c) In vitro transcription assays
The construction of pV4a.5 plasmids has been previously described (Sajjadi et al., 1987). The tRNA,V”’ gene containing 138 bp of 5’-flank was liberated by cleavage with EcoRI and HindHI. This 286-bp fragment was subcloned into pEMBL8(Dente et al., 1983) to create pV4a.5-138.
In vitro transcription assays were carried out as previously described (Sajjadi et al., 1987; St. Louis and Spiegelman, 1985). Each series of transcriptions contained ten input points ranging from 10 to 100 ng of template DNA and nonspecific DNA to a final concentration of 1 pg/50 ~1. Each mutant template was transcribed alongside a pV4a.5- 138 series which served as a control. The maximum level of transcription directed by each template (V,,,) was calculated by both a numerical and a graphical procedure (St. Louis and Spiegelman, 1985) and the
(b) In vitro mutagenesis Site-specific double-primer
mutagenesis was carried out by the method of Zdller and Smith (1984)
15
change in V,,, reported as a y0 increase or decrease over the transcription of the unmutated wild-type
phoretic
template
pV4a.5-138
carried
were repeated
out in parallel.
with two different
the Y0 changes in V,,, between the extracts.
All transcriptions S- 100 extracts
reported
and
as the average
Fig. 1A shows separation and
observed.
(a) A pentadeoxynucleotide of tRNAy
in the 5’4lanking region
gene determines the efficiency of tran-
scription The subclone of pV4a.5179 (Sajjadi et al., 1987), pV4a.5-138 was chosen for site-directed mutagenesis of the sequence TCGCT contained between nt -34 and -38 relative to the mature coding sequence. The mutants created and their transcription efficiencies relative to pV4a.5138 are listed in Table I. All mutant tamplates were transcribed in parallel with a set of pV4a.5138 DNA controls. Reactions contained between 10 and 100 ng of template DNA; the very low template input is an important technical consideration as previously shown by Wilson et al. (1985) and St. Louis and Spiegelman (1985). The Vmax values were derived from the intercept of a least-squares line of the cpm of transcript plotted vs. DNA input, as previously described (St. Louis and Spiegelman,
TABLE Change
1985).
of transcription the double
This product
products
mutation
from
nt -38(G);
had previously
been shown
at a G at nt -9 (Sajjadi
et al., 1987).
Fig. 1B shows a plot of the reciprocal
of the cpm of
transcript
AND DISCUSSION
of the electro-
-35(A). As shown in this figure, and in all other instances, only a single transcription product was to initiate
RESULTS
autoradiograms
vs. the reciprocal
of the DNA input which
allowed the derivation of V,,, values from the intercepts of the least-squares line describing the data. The data in Fig. 1B denlpnstrate
that the nucleotide
substitutions in the 5’-flanking DNA of pV4a.5-138 reduce the template efficiency significantly. Table I summarizes the average of the y0 change in V,,, values for each mutated template. Templates containing single nucleotide changes in the TCGCT sequence, i.e. at nt -34(T), -35(C) and -38(T) each resulted in approx. 30% decrease in V,,,. A double change at nt -35(C) and -38(T) resulted in a V,,, approx. 40% lower than wild type. In contrast, the single base substitution at nt -37(C) resulted in a small increase in V,,, relative to wild-type sequence. Furthermore, a change at nt -36(G) decreased transcription by only 1%. Together these latter two changes suggest that nt -37(C) and -36(G) are not important for transcription modulation (Sajjadi et al., 1987). The increase in V,,, seen with the change at nt -37(C) may be due to the increased A + T content of the 5’-flank. In contrast, the trans-
I in V,,,
Template
values obtained
from in vitro transcription
of TNNCT
mutants
a
relative
to pV4a.5-138
v max b y0 change
pV4a.5-138
TCGCT
(wild type)
GCGCT
[mutation,
nt -38(G)]
128.2
TCGAT
[mutation,
nt -35(A)]
TCGCA
[mutation,
nt -34(A)]
132 J32.8
GCGGT
[mutation,
nt -38(G);
-35(C)]
137.7
GCGAT
[mutation,
nt -38(G);
-35(A)]
TCTCT
[mutation,
nt -36(T)]
142 1 1.1*
TAGCT
[mutation,
nt -37(A)]
t12.6
a These point mutants
0 (control)
are described
in RESULTS
AND
DISCUSSION,
section a.
experiments for each of the mutants and is lJ VW was calculated from the data obtained from ten-point DNA input transcription expressed as y0 increase (upward arrows) or decrease (downward arrows) over the value or pV4a.5-138 transcribed in parallel. V,,,,, values are the average S-100 extract.
of determinations
using two different
S-100 extracts.
If,,,,, value with an asterisk
was calculated
using a single
16
A wild-type 2
1
3
4
5
6
-38G;-35A 7
8
9
10
Fig. 1. In vitro transcription pV4a.5-138
products,
[-38(G);
(B) Double reciprocal of product oftemplate
of pV4a.5138
and of the double mutant
respectively.
plot of transcription by Cerenkov
Transcriptions
-35A
products transcription
The data were plotted
DNA). The lines were derived by the method ofleast
and 0.999 for -38G,
at nt -38(G);
4
5
6
squares
-35(A).
(A) Autoradiogram
8
9
10
100
of a polyacrylamide
gel
of IO to 100 ng of pV4a.5-138
and
in MATERIALS
products
AND METHODS,
section c.
were excised (see panel A) and the amount
as l/V (V = cpm of transcript/h with correlation
7
75
from transcription
were at 24°C as described
data. Gel slices containing counting.
3
50
Lanes I-10 and 1 I-20 show the radiolabeled
-35(A)],
was determined
2
25
-25
of transcription
1
coefficients
of reaction)
vs. l/S (S = mass
of 0.998 for pV4a.5138
(triangles),
(squares).
version of a C to an A at nt -35 resulted in a 32% decrease in V,,,, emphasizing the specificity of the TCGCT sequence at nt -35(C). In our earlier paper (Sajjadi et al., 1987), we observed a more dramatic decrease in the level of transcription when the nucleotides 5’ to and ineluding TCGCT were removed and replaced by
vector sequences when compared to site-specific changes in TCGCT in Table I. The results presented here implicate the short nucleotide stretch immediately 5’ to TCGCT (to nt -45) as a possible secondary positive modulator of transcription as we previously suggested.
17
(b) Comparison
sequences of Droso-
of 5’-flanking
drop in transcription.
Together,
these data led us to
phila tRNA genes and their respective transcription
believe that a sequence with a general form TNNCT
efficiencies
would be a candidate
for further
examination.
We
therefore
searched the 5’-flanking region of other Drosophila tRNA genes for the presence of TNNCT
Data presented in Table I illustrate that nt -38(T), -35(C) and -34(T) are essential for maximal stimulation
of transcription
transversions nificantly
of pV4a.5-138,
at nt -37(C)
decrease
do not sig-
the rate of transcription.
on pArg (Schaack
and
TTTCT
TNNCT
between
transcribed.
Studies
S011, 1985) indicated
deletion of the sequence
TABLE
and -36(G)
sequences (Table II). Valine and arginine
whereas
tRNA
nt -30
In a previous
genes which have a
and -40
are efficiently
study, we discovered
that
a deletion endpoint pV4a.5-20R which acquired a TNNCT by a combination of vector and 5’-flanking
that
resulted in an 88 %
II
Correlation
of transcription
Template
il
efficiency Position
and the pentadeoxynucleotide
of TNNCT’
pYH48-Arg
- 38TTTCT
pEl.R-Arg
- 52TTGCT
- 40TATCT
d
- 88TCTCT
- 84TTTCT
- 69TTGCT
- 36TGCCT
d
pDt67R-Arg
- 15 ITAACT
pDt l7R-Arg
- 89TGACT
- 126TAGCT
in the 5’-flanking
region of a tRNA
Transcription’
Reference
+
Schaack
gene
et al. (1984)
+e +e
- 106TGTCT
- SOTTCCT - 53TCCCT
- 38TTGCT
- 33TTTCTd
p 11 F-Arg
No TNNCT
(to -40)
p35D-Arg
No TNNCT
pE3.8-Arg
No TNNCT
pl7D-Arg
No TNNCT
pAva4-Arg
No TNNCT“
-(+I -(+I
-(+P _
Dingermann
et al. (1982)
Dingermann
et al. (1982)
Dingermann
et al. (1982)
_e
pAsn8-Asn
- 42TGGCT
- 37TGGCT
pAsn6-Asn
No TNNCT
(to -48)
pAsnl-Asn
No TNNCT
(to -48)
pHis-His
- 46TTGCT
p48His- YHis
- 22TTGCT
pDt39R-:-Y”
- 36TACCT
pDt59R-;-Y” pDt5-Scr pDtlhH#
TNNCT
+
Lofquist
and Sharp
(1986)
and Sharp
(1986)
Lofquist
and Sharp
(1986)
- 38TTCGT
--(+I _ + -(+) + +
Lofquist
No TNNCT
+
St. Louis and Spiegelman
(1985)
+(-I +(-I + +
St. Louis and Spiegelman
(1985)
St. Louis and Spiegelman
(1985)
I-;;;
~ 39TAGCT
pDt 16H # 5-T;;
No TNNCT
pDt55-0.6-,V”’
- 39TGGCT
pV4a.5-179-T”’
- 38TCGCT
- 23TCTCT
pDt92R-,V”’
- 79TTACT
- 48TTGCT
Cooley et al. (1984) Cooley et al. (1984)
+(-I”
- 44TAACT
DeFranco
et al. (1982)
DeFranco
et al. (1982)
Rajput
et al. (1982)
Sajjadi
et al. (1987)
Addison
et al. (1982)
- 39TATCT pDt78R-:;’
No TNNCT
“ Designation
of recombinant
plasmids
or absence
of the positive
’ The presence Numbers
indicate
’ Symbols
the position
for transcription
- ( + ), inefficient is taken
c L. Duncan
containing
J. Leung,
and G.B.S.,
Drosophila tRNA
modulatory
of the 5’ T in the TNNCT effciences
-
TNNCT
sequence
in the 5’-flanking
, no detectable
and G.M. Tener, personal results.
transcription.
communication.
species is indicated
in the 5’-flanking
10% of pV4a.5-179);
the levels do not relate to pV4a.5-179. unpublished
genes. lsoacceptor
sequence
are: + , efficient (within
(less than 1 y0 of pV4a.5.179);
from the literature,
d C.H. Newton,
Leung et al. (1984) after the hyphen.
region of various
DNA, relative to the mature + (- ), inefficient
tRNA genes is shown. coding sequence
( + 1).
(less than 15% of pV4a.5-179);
For entries where evaluation
of transcription
efficiency
IS
sequences
at position
-20 relative to the gene was an
inefficient template when compared to pV4a.5179 (Sajjadi et al., 1987). This would seem to be similar to the pseudohistidine TNNCT
tRNA
gene
which
has
located at nt - 18 to -22 and transcribed
a at
other correspondences random frequencies.
which occurred
We would emphasize, TNNCT tRNA
that we do not believe that
is the only positive transcription genes.
As
at the above
discussed
in
effector for
RESULTS
AND
tRNAHi” gene on plasmid pHis (Cooley et al., 1984).
DIscussIoN, section a, our previous analysis suggested that nt -46 to -42 in pV4a.5-179 [AGTTG]
Plasmid
may
less than
1% of the efficiency pHis has a TNNCT
at nt -42 and -46 and
is an effective in vitro template. that of the tRNA,V”’ allogene This plasmid
is a moderately
of its counterpart
An unusua1 case is on plasmid
good template
and carries five copies of the TNNCT. tRNAA’g
gene (pDt17R)
was found
pDt92R. in vitro
Recently,
a
to transcribe
very efficiently with eight copies of TNNCT in its 5’-flank (C.H. Newton, J. Leung and G.M. Tener, personal communication). Genes coding for tRNAVa’ and tRNAArb which do not contain the TNNCT in their 5’-flank either fail to direct transcription or direct transcription very inefficiently in vitro. The TNNCT is also found in the 5’-flanking regions of other tRNA genes such as tRNAF (DeFranco et al., 1982) which directs efficient transcription and tRNAi’” and tRNALe” (Robinson and Davidson, 198 1) whose transcription has not been studied. It should be noted that two TNNCTs, as are found in the 5’-flank ofpV4a.5179, are not necessary to stimulate transcription, since phrg and other tRNA genes function as in vitro templates with only one copy. An important question raised by Table II is whether the occurrence of TNNCT is random in the 5’-flanking sequences of tRNA genes. The sequence does appear with expected random frequency (l/64) in the plasmid pBR322 (not shown). In a block of nt -25 to -45 relative to a tRNA gene, a single TNNCT sequence should occur with a Poisson frequency of 0.22. Of the 23 genes in Table II, 13/23 ( = 0.56) have a TNNCT in the nt -25 to -45 region while ten do not. Furthermore, when analysed as groups, of the genes which are transcribed at moderate to high efficiency, twelve of the 14 have TNNCT sequences in the nt -25 to -45 region. Conversely, of the genes which are transcribed at less than 1% of pV4a.5-179, none have a TNNCT sequence in the nt -25 to -45 region. Thus, the association of the sequence TNNCT with genes which are active templates is not random using our current data. We examined the 50 nt 5’ to the genes in Table II for other homologies and could find no
also
stimulate
transcription
1987). We have preliminary analysis
that
scription
of the tRNAs”
the same
G.B.S., unpublished
(Sajjadi
et al.,
evidence from a deletion sequence
stimulates
gene in pDt5 (F.G.S.
observations).
expect all genes with a TNNCT
tranand
Thus, we do not in their 5’-flanking
sequence to be transcribed equally well and there are genes without the TNNCT sequence which are effective in vitro templates. For example, pDt5 (Table II; St. Louis and Spiegelman, 1985), directs transcription equivalent to pV4a.5-179 in the absence of TNNCT. On the other hand, for one tRNASe’ allogene (pDt 16H No. 1; St. Louis and Spiegelman, 1985), a deletion mutation which removed .5’-Banking sequences distal to nt -36 disrupted a TNNCT and resulted in a four-fold decrease in V,,,. In addition, it may be that pDt5 would serve as a more efficient template if a TNNCT were placed between nt -25 and -45 in its 5’-flanking region. It does appear that multiple TNNCT sequences do not provide additional stimulation of transcription since pDt92R-Val, is not a better template than pV4a.5179, but has more copies of the TNNCT sequence. Experiments investigating the mechanism of the effect of TNNCT on transcription are in progress. Finally, other examples of 5’-flanking sequences which affect transcription of class-III genes have been found in other lower eukaryotes (Larson et al., 1983; Raymond and Johnson, 1983; Shaw and Olson, 1984). These sequences have no homology to TNNCT. Similar kinds of sequences have yet to be identified in higher eukaryotes although given the complexity of the class-III gene family and of the transcription apparatus for these genes, we would expect such sequences to exist.
ACKNOWLEDGEMENTS
We would like to thank Loverne Duncan for the preparation of S-100 extracts, David Goodin for the
19
gift of Escherichia coli BW313 and helpful suggestions on site-directed mutagenesis. This work was supported by Medical Research Council of Canada Grant 68-7364.
Rajput,
B., Duncan,
L., DeMille,
D., Miller
Jr.,
R.C.
and
Spiegelman, G.B.: Transcription of cloned transfer RNA genes from Drosophila melanogaster in a homologous cell-free extract.
Nucl. Acids Res. 10 (1982) 6541-6550.
Raymond,
G.R. and Johnson,
sequences
J.D.: The role of non-coding
in transcription
and processing
DNA
of a yeast tRNA.
Nucl. Acids Res. 11 (1983) 5969-5988. Robinson,
R.R. and
sequences. Addison,
W.R.,
Hayashi,
Astell,
C.R.,
Delaney,
A.D.,
S., Miller Jr., R.C., Rajput,
Gillam,
I.C.,
B., Smith, M., Taylor,
D.M. and Tener, G.M.: The structures
G., Castagnoli,
L., Melton,
of a eukaryotic
noncontiguous
D.A.
tRNAPro
Cortese,
R.:
gene is composed
and
of
regions. Proc. Natl. Acad. Sci. USA 79 (1982)
Cooley, L., Schaack,
J., Johnson-Burke,
D.: Transcription pseudogene. DeFranco, R.C.
factor binding
D., Thomas, gene
and
a
Siill, D.: Genes
tRNA”‘$
S., Tener, G.M., Miller Jr., Drosophila
melunogaster. Nucl. Acids Res. 10 (1982) 5799-5808. Dente,
L., Cesarini,
of single
G. and Cortese,
R.: pEMBL:
plasmids.
Acids
stranded
Nucl.
Res.
T., Johnson-Burke,
genes control extract. Kunkel,
D., Sharp,
11 (1983)
S., Schaack,
J. and
of Drosophila tRNAArs
sequences
their in vitro transcription
in Drosophila cell
without
Rapid
and
phenotype
efficient
selection.
site-specific
Proc. Natl. Acad. Sci. USA 82
A short 5’-flanking Acad.
J., Young, L.S. and Sprague,
region containing
for silkworm
conserved
K.U.:
sequences
is
alanine tRNA gene activity. Proc. Nat].
W.R., Delaney,
Jr., R.C., Spiegelman,
A.D., MacKay,
G.B., Grigliatti,
R.M., Miller
T.A. and Tener, G.M.:
Drosophila melanoguster tRNAY,“’ genes and their allogenes. A. and
Drosophila
Sharp,
melanogaster
RNA polymerase
S.: The tRNA:“”
5’-flanking
sequences
genes differentially
III. J. Biol. Chem. 261(1986)
with
S., Dingermann,
T., Johnson-Burke,
5’- and
3’-flanking
sequence
and stable complex
dependence
formation.
D., tRNA
for tran-
J. Biol. Chem. 259
(1984) 1461-1467. of a Drosophila tRNAArs
J. and Sol], D.: Transcription
gene in yeast extract: transcription Sharp,
5’-flanking
sequence
dependence
for
system. Nucl. Acids Res. 13
in a heterologous
S., Dingermann,
T., Schaack,
D.: Transcription
J., DeFranco,
of eukaryotic
tRNA
D. and Still,
genes
of control regions using a competition
in vitro,
I.
assay. J. Biol.
Chem. 258 (1983) 2440-2446. Shaw,
K.J. and
sequences
Olson,
M.V.:
Effects
of altered
5’-flanking
of a Saccharomyces
on the in vivo expression
K.U., Larson,
signals required
of
arrest
14600-14606.
D. and Morton,
D.: 5’-flanking
for activity of silk-worm in vitro transcription
sequence
alanine tRNA genes
systems.
Cell 22 (1980)
171-178. St. Louis, D. and Spiegelman, of cloned tRNAs”’
region
G.B.: Steady-state
kinetic analysis
genes from Drosophila melanogasier. Eur.
148 (1985) 305-313.
Wilson, E.T., Larson, controls
D., Young, L.S. and Sprague, tRNA
gene transcription.
K.U.: A large
J. Mol. Biol. 183
(1985) 153-163. Ziiller,
Gene 34 (1984) 207-217. Lofquist,
gene.
J. Biochem.
Sci. USA 80 (1983) 3416-3420.
Leung, J., Addison,
A.R.: DNA seqencing
Proc. Nat]. Acad. Sci. USA 74
L. and Siill, D.: The extent of a eukaryotic
in homologous
D., Bradford-Wilcox,
required
inhibitors.
J., Sharp,
Sprague,
mutagenesis
(1985) 488-492. Larson,
S. and Coulson,
crrevisiae tRNATy’ gene. Mol. Cell. Biol. 4 (1984) 657-665.
J. Biol. Chem. 257 (1982) 14 738-14 744. T.A.:
its transcription
206 (1987) 279-284.
(1977) 5463-5467. Schaack,
Analysis
Siill, D.: The 5’-flanking
G.B.: Identiti-
region of a Drosophila
in the 5’-flanking
(1985) 2803-2814.
a new family
1645-1655. Dingermann,
F., Nicklen,
chain-terminating
Schaack,
for tRNAkYS from
Miller Jr., R.C. and Spiegelman,
in vitro. Mol. Gen. Genet.
scription
Mol. Cell. Bio. 4 (1984) 2714-2722.
D., Burke, K.B., Hayashi, and
B. and Soll,
is limited by the 5’-flanking
of Drosophila tRNA”‘”
regions
F.G.,
Cooley,
1195-1199.
intervening
Cell 23 (198 1) 25 l-259.
cation of sequences
Sanger,
251 (1982) 670-673. Promoter
Sajjadi,
of a Drosophila
D.: Analysis
two tRNALe” genes contain
melanogaster tRNA,V”’ gene that modulate
of genes hybridizing
from Drosophila melanoguster. J. Biol. Chem.
with tRNAy”’ Ciliberto,
Davidson,
tRNA gene cluster:
REFERENCES
M.J. and
genesis: and
Smith,
M.: Oligonucleotide
a simple method
a single-stranded
479-488. Communicated
by S.T. Case.
directed
using two oligonucleotide DNA
template.
DNA
mutaprimers
3 (1984)