From the Boyce Thompson Institute for Plant Research
and the § Section of Genetics and Development, Cornell
University, Ithaca, New York 14853-1801
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ABSTRACT |
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Plant mitochondrial genes are often transcribed
into complex sets of RNAs, resulting from multiple initiation sites and
processing steps. To elucidate the role of initiation in generating the
more than 10 cox2 transcripts found in maize mitochondria,
we surveyed sequences upstream of cox2 for active
promoters. Because the cox2 coding region is immediately
downstream of a 0.7-kb recombination repeat, cox2 is under
the control of two different sets of potential expression signals.
Using an in vitro transcription assay, we localized four
promoters upstream of the coding region in the so-called master
chromosome, and two promoters upstream of the coding region in the
recombinant subgenome. Ribonuclease protection analysis of labeled
primary transcripts confirmed that all but one of these promoters is
active in vivo. Primer extension was used to identify the
promoter sequences and initiation sites, which agree with the consensus
established earlier for maize mitochondria. This study identified two
unusual promoters, the core sequences of which were composed entirely
of adenines and thymines, and one of which was a complex promoter
consisting of seven overlapping units. Deletion mutagenesis of the
complex promoter suggested that each of its units was recognized
independently by RNA polymerase. While each active promoter fit the
maize core consensus sequence YRTAT, not all such sequences surveyed
supported initiation. We conclude that in vitro
transcription is a powerful tool for locating mitochondrial promoters
and that, in the case of cox2, promoter multiplicity
contributes strongly to transcript complexity.
The transcriptional strategies of vascular plant mitochondrial
genomes differ from those of their fungal and metazoan counterparts, with a major region being genome structure. Plant mitochondrial genomes
are relatively large and the genes are mostly dispersed (1, 2). In
contrast, metazoan genomes are compact and have a single promoter for
each DNA strand, and the Saccharomyces cerevisiae mitochondrial genome is 75 kb,1 and contains about 20 promoters (reviewed in Ref. 3). The maize mitochondrial genome, the
expression of which we have studied, can be mapped as a 570-kb circle
with numerous repeated elements mediating recombination, leading to a
complex set of overlapping molecules (4). Based on recent microscopic
studies (5), these molecules are likely to be linearly permuted.
Promoter analysis in plant mitochondria has been accelerated in the
last several years by the development of in vitro
transcription systems from wheat (6), maize (7), and pea (8). Extensive mutational analysis of the maize atpA and cox3
promoters showed that the only universally-present sequence required
for transcription initiation in vitro was YRTAT (Y = T
or C and R = G or A), located at or immediately upstream of the
start site (9, 10). Even this degenerate sequence, however, is not
found near all 5' termini identified as transcription start sites based
on their ability to be capped in vitro by guanylyl
transferase (11). While some of these promoters may be recognized by
specific transcription factors (12), the frequency of active promoters
has not been surveyed over large regions of any plant mitochondrial
genome. Such an approach might aid in identifying contextual features other than YRTAT that constitute functional promoters, at least in
maize mitochondria.
Maize cox2, which encodes subunit II of cytochrome
c oxidase, was the first plant mitochondrial gene to be
cloned and sequenced (13). RNA filter hybridization analysis of
cox2 transcripts in maize mitochondria identified at least
11 distinct messages. Since cox2 contains only a single
intron, multiple 5' and/or 3' ends must account for most of these RNA
species. Here we investigate how genomic context and promoter
multiplicity contribute to cox2 transcript diversity, and
show that in vitro transcription is a useful means to
identify previously unknown promoters.
Plasmid and Cosmid Clones--
Cosmids N5G8 and N6A6 (Fig. 1;
Ref. 4) were obtained from Dr. Christiane Fauron (University of Utah).
The BamHI and XhoI fragments illustrated in Fig.
2 were gel-purified and inserted into plasmid vectors, generating
plasmids pN1, pN2, pN4, and pN7. To generate antisense single-stranded
DNA for mapping of capped transcript 5' termini, the subfragments shown
in Fig. 2 were inserted into pBluescript (Stratagene, Inc.) in the
appropriate orientation. These plasmids were pN1VV (from the
EcoRV site in pBS to the EcoRV site in pN1),
pN2AR (from the XbaI to RsaI sites in pN2), and pN7PT (from the ApaI site in pBS to the AatII
site in pN7).
Oligonucleotides SL5 and SL6 (region B) and SL7B and SL9 (region A)
were designed based on unpublished sequences provided by Dr. D. Lonsdale (John Innes Institute, Norwich, United Kingdom) or on sequence
data that we generated from the region A cosmid, respectively. SL5
begins 804 bp upstream of the 0.7-kb repeat and has the sequence
ACTCAGTCCTGCTAG, and SL6 begins 163 bp upstream of the repeat and has
the sequence GATGTAACGAGCACTC. SL7B begins approximately 3190 bp
upstream of the repeat and immediately upstream of the EcoRV
site, and has the sequence CAACATGAGAAGATC. SL9 begins 10 bp upstream
of the repeat and has the sequence ACAGCGTTTGTCCAT.
To generate the deletions of the complex promoter illustrated in Fig.
7, pN2 was linearized with MluI, which cleaves immediately upstream of promoter A2a. 10 µg of plasmid DNA were digested with 5 units of Bal31 for 10-45 s at room temperature. The pooled
products were repaired with the Klenow fragment of DNA polymerase and
religated. Escherichia coli colonies were screened by the
polymerase chain reaction for inserts of the approximate size desired,
and the precise deletion end points were determined by DNA sequencing.
In Vitro Transcription--
Subclones of the cosmids were
linearized with various restriction enzymes as shown in Fig. 3, and
in vitro transcription was carried out as described
previously (7). The sizes of transcripts were estimated by comparison
to a DNA size ladder made using 32P-labeled Capping of mtRNA and Primer Extension Analysis--
RNA was
isolated from gradient-purified mitochondria of 4-day-old dark-grown
maize seedlings. 100 µg of RNA was capped using 15 units of guanylyl
transferase and 250 µCi of [32P]GTP, as described
previously (11). 10 µg (106 cpm) of labeled RNA was
incubated with 500 ng of single-stranded DNA (ssDNA) under conditions
previously described (11), with denaturation at 85 °C for 10 min and
annealing at 42 °C overnight. The hybrids were digested with 50 or
250 ng of RNase A. The protected transcripts were separated in
denaturing polyacrylamide gels using the DNA markers described above.
Primer extension was carried out using the same RNA preparations and
oligonucleotides labeled with [ Recombination Across the 0.7-kb Repeat Places the cox2 Coding
Region Downstream of Two Different Sets of Potential Expression
Signals--
Fig. 1 shows that the
cox2 coding region is located immediately downstream of a
0.7-kb direct repeat sequence, present in two copies in the maize
mitochondrial master chromosome. Recombination across these repeats
generates the two postulated subgenomes shown at the right of Fig. 1.
In the master chromosome, cox2 is preceded by sequences
which we have designated region B, whereas in subgenome 1, cox2 is downstream of region A. Conversely, in the master
chromosome region A is upstream of sequences of unknown function
(designated NC), whereas region B is upstream of NC in subgenome
2.
Since the master chromosome and subgenomes co-exist in vivo
(4),2 promoters driving
cox2 transcription could be located in regions A and B,
and/or in the repeat sequence. Fig. 2
shows a more detailed map of the A, B, cox2, repeat and NC
regions, and the plasmid clones that were made in order to
systematically search these regions for promoters using in
vitro transcription. While most of region B, the repeat, and the
cox2 coding sequence are present in the data base, region A
is not, and therefore clones were mapped for restriction endonuclease
cleavage sites. Closely spaced sites were required because our in
vitro transcription assay best resolves 50-500-nt transcripts,
thus our goal was to find unique sites 0.5 kb apart or less. Clones
linearized at each unique site were used as templates for in
vitro transcription.
Fig. 3 (top) shows examples of
in vitro transcription reactions in which promoter activity
could be identified. Other plasmid/restriction endonuclease
combinations did not yield resolvable transcripts. A summary of the
clones tested and the transcripts obtained is given in Table
I. Fig. 3 (bottom) summarizes
the locations of promoters deduced from in vitro
transcription. Two promoters are found in region A: A1, which is
located approximately 3 kb upstream of the 0.7-kb repeat; and A2, which
is immediately upstream of the repeat. Remarkably, A2 (hereafter
referred to as the "complex promoter") gave rise to seven
transcripts differing in size by 5 nt. Four promoters were found in
region B, all clustered within 1 kb of the 0.7-kb repeat. No promoters
were detected within the repeat. Taken together, these results
suggested that cox2 could be transcribed from either the
master chromosome or subgenome 1, and that up to six promoters,
including a complex promoter, might contribute to RNA accumulation
in vivo.
Five of the Six Promoters Identified in Vitro Are Active in
Vivo--
It was possible that in vitro transcription would
give artifactual promoter activity at a site that was not used in
vivo. To determine whether the in vitro start sites
coincided with primary transcript 5' ends, we used a combination of
in vitro guanylyl transferase capping (G-capping) and RNase
protection. Since plant mitochondrial transcripts are not capped
in vivo, primary transcripts (those with di- or triphosphate
termini) can be labeled in vitro with
[32P]GTP. Total maize mitochondrial RNA was labeled in
this way and then annealed to ssDNA complementary to the
cox2 regions containing putative promoters. The hybrids were
digested with RNase A and analyzed by gel electrophoresis.
Fig. 4A shows the protected
primary transcripts corresponding to promoters A1, A2, B1, B3, and B4.
The promoter identifications were based on the sizes of protected
transcripts, coupled with knowledge of the in vitro start
sites (Fig. 3) and primer extension (see below). Fig. 4B
summarizes the verified promoters and the ssDNAs used to protect the
primary transcripts. Of the promoters identified by in vitro
transcription, only B2 was not identified by capping. This suggests
that either B2 is an in vitro artifact, or that the primary
transcript from B2 is rapidly processed in vivo. Although we
cannot distinguish between these two alternatives, the weak usage of B2
in vitro (Fig. 3) suggests that at best, B2 is a marginal
promoter in vivo. Taken together, these results confirm that
cox2 is transcribed by multiple promoters in two distant
genomic regions, and that the maize mitochondrial in vitro transcription system can be reliably used to locate promoters in
sequences of unknown function.
Each cox2 Promoter Fits the Previously Established
Consensus--
We have previously determined the essential features of
maize mitochondrial promoters by using several types of mutagenesis (7,
9, 10). A key feature of these promoters is the so-called core
sequence, which has the consensus YRTAT; mutations in the core severely
reduce or abolish transcription in vitro. Transcription initiates at the final T or within a few bp downstream. Primer extension was used to identify the precise initiation sites of the
cox2 promoters. These experiments were carried out in using total mtRNA, and the results are shown in Fig.
5. For each promoter identified by
capping and in vitro transcription, a single start site was
found, except that seven start sites were found in the complex promoter
(A2a-g).
The promoter sequences and initiation sites are summarized in Fig.
6, and compared with the consensus
sequence derived for the atpA promoter (9). All of the
promoters had sequences within 5 bp upstream of the start site that
fully matched the consensus sequence for the core region, either CGTAT
or TATAT. Their match with the overall consensus sequence was variable,
ranging between 79% and 100%. The importance of the consensus
sequence apart from the core is suggested by the fact that promoter A1,
which was the weakest promoter in vitro, had the lowest
percent match. Interestingly, promoters A2b-g and B3 are the first
mitochondrial promoters identified in which the core sequences are
composed of only A and T nucleotides.
Deletion Mutagenesis Suggests That the Complex Promoter Is
Recognized Stochastically--
The complex promoter A2 is highly
unusual and consists of one "typical" CGTAT core sequence promoter,
followed by seven copies of the repeated sequence GTATATA. This
repeated sequence contains a putative TATAT core sequence, and gives
rise to six additional overlapping promoters, with the overlap coming
between the core and the purine-rich upstream domain (see Fig. 6).
We envisioned two models for recognition of the complex promoter by the
transcriptional machinery, as diagrammed in Fig.
7C. In the scanning model, the
first (CGTAT core) promoter nucleates the transcriptional apparatus,
which then either initiates transcription or scans downstream sequences
for an alternative promoter. In the stochastic model, the transcription
apparatus can independently recognize each individual promoter,
although since the mitochondrial genome is multipartite this could be
occurring on separate molecules. The RNase protection and primer
extension data (Figs. 4 and 5) show that approximately twice as many
transcripts initiate at the first promoter as at each of the downstream
promoters, which yield similar amounts of transcripts. These results
could favor the stochastic model, if one assumes that CGTAT is a
stronger core sequence than TATAT, or if one assumes that the
downstream promoters are disfavored because they are overlapping.
However, a scanning model also predicts increased initiation at the
first promoter, but also a successive reduction in initiation at each following promoter, which is seen in Fig. 3 (in vitro) but
not in Figs. 4 and 5 (in vivo).
To determine whether the first promoter was required to anchor
transcription as proposed by the scanning model, we generated Bal31 deletion mutants lacking the first promoter and
variable amounts of the downstream promoters, as shown in Fig.
7A. These constructs were used for in vitro
transcription analysis, as shown in Fig. 7B. In each case,
the remaining promoters are transcriptionally active, which rules out a
scanning model dependent on the CGTAT-containing promoter. In the
analysis of these deletion mutants, we noted that there was some
ambiguity in the assignment of start sites. In particular, in the
WT lane there is an additional diffuse band migrating slower than the band that we believe represents promoter A2a,
and in the deletion constructs the number of residual start sites did
not always coincide with the expected number. For example, we expected
two additional start sites in mutant Here we have looked globally at promoters potentially driving
maize mitochondrial cox2 transcription. The screen for
promoters was carried out using an in vitro transcription
system, and the reliability of this approach was confirmed in five of
six cases using analysis of mtRNA. Each promoter conforms to a
previously established consensus sequence for maize mitochondrial
promoters, although there are some with unusual features. This promoter
multiplicity can help account for the complexity of accumulating
cox2 transcripts.
Approximately 8.4 kb of DNA was examined for promoter activity in this
study. Of the six promoters active in vitro, each was flanked by a sequence fitting the consensus YRTAT, with little homology
upstream (see Fig. 6). Clearly, if YRTAT were the only requirement for
promoter activity, a much larger number should have been detected. For
example, in the part of region B that we surveyed (4.75 kb), YRTAT
appears 18 times, including 5 matches of CGTAT, the most common core
motif in maize mitochondrial promoters (11). The number of YRTAT
matches is close to that expected by chance, suggesting that there is
no bias to exclude such sequences from the genome. These results
suggest that YRTAT is necessary, but not sufficient for promoter
activity in our assay system. It is possible that other features such
as DNA bending, which occurs at human and yeast mitochondrial promoters
(14, 15), contribute to promoter recognition and usage in maize mitochondria.
Promoter B2 was active in vitro but not in vivo.
While this result could be explained by a rapid processing of
B2-initiated transcripts in vivo, it is also possible that
this promoter-like sequence is not recognized in the cellular
environment. For example, a conserved sequence that acts as an 18 S
rRNA promoter in Arabidopsis, potato, and
Oenothera is inactive in vivo in pea (16). This again points to features other than the primary sequence that mediate
promoter site selection. Interestingly, the yeast mitochondrial genome
also contains many promoter-like sequences that are not active in
vivo, but are active in vitro (17). Analysis of these variants suggests that they are inactive in vivo not because
RNA polymerase is unable to bind, but instead because elongation does not occur. Thus, multiple criteria must be satisfied if transcription initiation is to occur successfully.
Some of the promoters identified in this study are far from the
cox2 coding region, such as A1 (>4 kb) and B1 (>1.5 kb).
The fact that these are used for cox2 transcription in
vivo is consistent with the lengths of some cox2
transcripts (13) as well as reverse transcription-polymerase chain
reaction results in which cox2-primed cDNAs could be
amplified with primers downstream of each promoter.2 While
such a distance between coding region and promoter is unusual, it is
not unprecedented. For example, the atpA promoter is more than 2 kb upstream of the coding region, and there may be another promoter even further upstream (11). Furthermore, several other maize
mitochondrial genes have multiple promoters (18, 19). So far, however,
the combination of promoter distance and number, along with the
presence of the coding region downstream of a recombination repeat,
places cox2 in a unique category.
The 0.7-kb repeat upstream of cox2 contains no promoters,
and does not flank the cox2 gene in other maize cytoplasms
such as cms-T and NA188 (1). Thus, the configuration in the genome we
examined (NB37) is the only one of the three in which repeat-driven transcription could express cox2. In the maize ancestor
teosinte, the cox2 transcription pattern is simple (20) and
driven by two promoters, one of which is dependent on a nuclear locus
that may encode a specific transcription factor (12). These data highlight the plasticity of plant mitochondrial genomes, and the multiplicity of promoters may ensure the expression of genes despite frequent mtDNA rearrangements during evolution (21).
Two unusual promoters were uncovered by this study, namely A2 and B3.
Both of these promoters have the novel core consensus TATAT, except
A2a, which has a classic core motif. While we have not tested the
importance of each of these positions in the cox2 promoters
by mutagenesis, there is no other nearby sequence that matches the
consensus, and indeed more than 10 bp upstream of promoter B3 consist
of A or T. The ability of all-A+T sequences to function as
mitochondrial promoters is illustrated indirectly by the recovery of
two yeast petite mutants which are able to replicate, yet possess
mitochondrial genomes consisting only of A-T base pairs (22). Since
replication is most likely initiated with an RNA primer (23), one can
infer that transcription initiation occurs within these A-T sequences.
Promoter A2 consists of seven units punctuated by guanosines, and these
units overlap if one includes the upstream purine-rich region. Each of
these units can be recognized both in vitro and in
vivo, which is most consistent with them acting independently. Our
results do not address the question of whether more than one transcription complex can simultaneously bind to this region. This
complex promoter could have arisen by slippage during DNA replication,
a mechanism that probably can generate short tandem repeats in
chloroplasts (24). Overlapping promoters are somewhat unusual in the
literature, and we have not found anything closely resembling
cox2 promoter A2. There are related instances, for example
the interspersed -35 and -10 elements of the two E. coli valyl-tRNA synthetase promoters (25), and the spinach chloroplast 16 S
rRNA nuclear-encoded RNA polymerase (NEP) promoter, which lies between
the -35 and -10 elements of an E. coli-like promoter (26).
While the valS promoters are used differentially in
vivo and in vitro, in vivo and in
vitro assays for spinach rrn16 and cox2 are
in agreement. Whether A2 has any special significance for
cox2 expression in vivo remains to be determined.
In summary, promoter multiplicity is not uncommon in
mitochondria, and seems to be particularly prevalent in maize. One
could speculate that this arrangement lends flexibility to modes of gene expression, particularly if promoters are differentially used and
if the resulting different 5'-untranslated regions influence translational yield. Maize mitochondrial transcripts (27) and translation products (28) have been shown to vary between tissues, consistent with this possibility. Another reason to maintain multiple promoters is to maintain gene expression in the face of frequent genome
rearrangements, including intragenomic recombination, as discussed
above. However, in vitro experiments suggest that
transcription termination may not be efficient in plant mitochondria
(29), meaning that read-through synthesis along with RNA processing events could substitute in some cases for gene-specific promoters, as
is the case for some chloroplast genes (30). Evidence for transcription
of known non-coding regions in maize mitochondria (31) points to RNA
stability as another key control point in gene expression. A
contrasting point of view to the above is that complex transcription
patterns are a consequence of multiple promoters, which themselves are
a consequence of a promiscuous transcriptional machinery.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
X174 RF DNA
digested with HinfI.
-32P]ATP. Sequence
ladders were generated from appropriate plasmid clones using the same primers.
RESULTS
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Fig. 1.
Recombination across a 0.7-kb repeat places
the cox2 coding region downstream of two different
sets of potential expression signals. The circle at the
left represents the full genetic complexity of maize mtDNA
(570 kb). The positions of the cox2 coding region, the
0.7-kb repeat discussed under "Results," upstream regions
arbitrarily labeled A and B, and a region of
unknown function (NC, non-coding) are shown, but are not to
scale. N5G8 and N6A6 refer to cosmid clones from which the plasmids
shown in Fig. 2 were derived. Recombination across the 0.7-kb direct
repeats generates the postulated 550-kb and 20-kb subgenomes shown at
right. DNA filter hybridizations clearly show that products
of recombination across the 0.7-kb repeats exist in vivo
(see Footnote 2).
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Fig. 2.
Maize mtDNA regions upstream of the
cox2 gene. N6A6 and N5G8 are cosmid clones shown
in Fig. 1, which represent segments of the master chromosome. These
served as the sources of the plasmid clones indicated beneath each
panel (see "Experimental Procedures"; plasmids represented by
arrows were used to produce the ssDNAs used in the
experiments shown in Fig. 4). The 0.7-kb repeats are represented by
gray arrows. The arrowheads marked
SL7B, SL9, SL5, and SL6 refer to the oligonucleotides used for primer
extension. Relevant restriction endonuclease sites shown are:
A, XbaI; B, BamHI;
C, AccI; G, BglII;
N, NsiI; R, RsaI;
S, SpeI; T, AatII;
U, StuI; V, EcoRV;
X, XhoI. A 1-kb scale is shown near the
top on the right side.
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Fig. 3.
Mapping of cox2 promoters
using in vitro transcription. The top
panels show in vitro transcription products for
each of the active templates, which are labeled by the number of the
subclone and the restriction endonuclease used to linearize it
(e.g. 7T is the N7 subclone linearized at the
AatII site). The transcript size in kb and promoter
identified are indicated at the left for each set of lanes.
The bottom panels are maps of the regions
searched for promoters for cox2, templates used, transcripts
obtained, and promoters identified. The promoters are shown as
bent arrows and are labeled based on the region
in which they are found and order in which they occur, and the 0.7-kb
repeats are represented by gray arrows. See the
legend to Fig. 2 and Table I for additional details.
In vitro transcription templates and their products
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Fig. 4.
Identification of cox2
primary transcripts by G-capping. A, results of
RNase protection of guanylyl-transferase-labeled primary mitochondrial
transcripts by the ssDNAs indicated at the top of each
panel. 250 or 50 ng of RNase A were used in each experiment.
HinfI-digested X174 markers are in the left
lanes of each panel and approximate sizes (in nt) are shown
at the left. The promoters verified by the protected primary
transcripts in each panel are indicated at the right.
B, a diagram summarizing the results. The promoters are
shown as bent arrows, the 0.7-kb repeats are
represented by gray arrows, and the
arrowed lines below each diagram represent the
ssDNAs used for RNase protection. See the legend to Fig. 2 for
additional details.
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Fig. 5.
Identification of transcription initiation
sites using primer extension. End-labeled primers were used for
extension of mitochondrial RNA and sequencing of cloned templates, and
fractionated side by side in denaturing polyacrylamide gels. The
primers used are indicated in Fig. 2. The core promoter sequence is
bracketed on the left, and details are given in
Fig. 6. Note that the order of lanes is different for the gel showing
the location of promoter B1.
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Fig. 6.
Transcription initiation regions for
cox2. Numbering at the top
is with reference to the consensus sequence derived from analysis of
atpA, as described in the Introduction, where +1 is the
transcription initiation site. The columns marked "txn start" and
"promoter" refer to the initiation site relative to the +1 position
of the atpA-based consensus sequence and the cox2
promoter identifier, respectively. Bases not matching the consensus are
shown in lowercase, and the boxes containing them
are shaded. In the consensus sequence, R = G or A, Y = C or
T, N = no base preference, and "?" indicates that the position
was not tested by mutagenesis (9).
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Fig. 7.
Deletion mutagenesis of the complex promoter
A2. A, sequences of the wild-type complex promoter and
its deletion derivatives. The promoters are shown as bent
arrows with the CGTAT core promoter in black and
the others in gray. The CGTAT core and one of the repeat
units are underlined in the WT sequence. B,
in vitro transcription results for WT and deletion mutants
1-
5. The transcripts are labeled with the corresponding promoter
from which they originate. An alternative interpretation is that the
diffuse band above that labeled "a" is in fact the A2a transcript
from WT, which would imply that the band labeled "g" is an
artifact. The identification of bands in this experiment for the WT
construct was based on its resemblance to earlier experiments, such as
the one shown in Fig. 3. C, two models for complex promoter
recognition, as discussed under "Results." RNA polymerase is
represented as having two subunits, based on the known characteristics
of the yeast mitochondrial enzyme.
3 versus
5, but
if this is the case the two largest transcripts from
3 were unexpectedly weak. These subtleties may result from sequence contexts arising out of the ligation of different bidirectional deletions.
DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank Dr. Christiane Fauron for providing the cosmid DNAs used in this study, and Dr. David Lonsdale for unpublished maize mtDNA sequences.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant R01GM52560 (to D. B. S.) and a National Institutes of Health predoctoral training grant (to D. S. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Boyce Thompson Institute, Tower Rd., Ithaca, NY 14853. Tel.: 607-254-1306; Fax: 607-255-6695; E-mail: ds28{at}cornell.edu.
The abbreviations used are: kb, kilobase pair(s); bp, base pair(s); nt, nucleotide(s); ssDNA, single-stranded DNA; WT, wild-type.
2 D. S. Lupold and D. B. Stern, unpublished results.
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REFERENCES |
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