Download Sequence and transcription analysis of the Petunia mitochondrial

Volume 14 Number 20 1986
Nucleic Acids Research
Sequence and transcription analysis of the Petunia mitochondria! gene for the ATP synthase
proteolipid subunit
EUora G.Young, Maureen R.Hanson1* and Peter M.Dierks
Department of Biology, University of Virginia, Gilmer Hall, Chariottesville, VA 22903 and 'Section
of Genetics and Development, Cornell University, Bradfield Hall, Ithaca, NY 14853, USA
Received 12 June 1986; Accepted 11 September 1986
ABSTRACT
He have sequenoed the Petunia hybrlda gene that specifies the
proteolipid subunit of the mitochondrial F o ATP synthase and have used this
gene to investigate plant mitochondrial gene transcription. The Petunia
atp 9 gene contains a single open-reading frame capable of specifying a 77
amino aoid-polypeptide that is homologous to bovine, fungal and maize
proteolipid subunits. S1 protection identified 3 transcripts in a ratio of
1:5:100 in the Petunia tissues tested. The transcripts share a common 3'
terminus but have 5' termini that map 528, 266, and 121 nucleotides
upstream of the translation start site. The 5' terminus of the longest
transcript maps to the sequenoe ATATAGTA, which is nearly identical to the
yeast mitochondrial transcription initiation site ATATAAGTA. Primer
extension analysis indicates that these two shorter transcripts are not due
to splioing. The two shorter transcripts originate at sequences homologous
to sites at 5' termini of two pea and maize genes. These consensus
sequences may signal processing events other than splicing.
INTRODOCTIOH
The location for the gene for the proteolipid subunit of mitochondrial
ATP synthase varies In different organisms.
In mammals, a nuolear-enooded
subunit is synthesized on cytoplasmic ribosomes and imported into the
mitochondrion1.
In Saooharomyces cerevlslae and maize, this subunit Is
specified and made in the mitoohondrlon^-11.
Heuroapora crassa is unusual
In that both the nuoleus and the mitochondrion contain a gene for ths
proteolipid subunit^.
However, only a nuclear transcript and a
oytoplasmically-synthesized subunit have been identified^.
Here we report the isolation and sequencing of an ATP synthase
proteolipid subunit gene from the mitoohondrial genome of Petunia hybrlda
line 3704. We also describe the transcripts of this gene by S1 nuclease
protection and primer extension analysis.
Northern analyses reported for a
number of sequenced plant mitochondrial genes3,6-9 have not defined the
nature of their transcripts.
Analysis of yeast mitochondrial genes has
allowed the identification of sequences directing the site of transcript
C IR L Pren Limited, Oxford, England.
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initiation10"12, sequences affecting promoter 3trength813-1t,
sequences signalling RHA processing15.
an(j
consensus
Comparisons of the transcript maps
of the Petunia gene described here with those of yeast and other plant
nitochondrial genes3,6,8,9,12,16,17 reveal three different short conserved
sequences at the 5' termini.
We propose that one is involved in transcript
initiation and that the other two conserved sequences signal site3 for RNA
processing.
HATERIALS AND METHODS
Plant Material
Petunia lines 3704, 3688, and 3699 were obtained from S. Izhar18.
Line 3704 carries a P. hybrlda nucleus and cytoplasm.
both contain the £. parodli nuclear genomes.
Lines 3688 and 3699
Line 3688 carries the Petunia
oytoplasmic male sterile (CMS) cytoplasm and line 3699 oarries the normal
fertile P. parodli cytoplasm.
Huolelo a d d isolation, Southern and sequence analysis
Petunia leaf total DNA and Petunia tissue RNA was isolated as
described elsewhere1^.
Mitochondria and mitochondrial DNA was isolated
aocording to Hanson e_t al..20, except a KI gradient was used for RNA
banding21.
A few samples of total leaf RNA were purified over oligo dT
cellulose Type 2 (Collaborative Research).
DNAs were digested with restriction enzymes under standard conditions,
electrophoresed on agarose gels, and transferred to nitrocellulose
aocording to Thomas22.
Restriction fragments were eluted from low gelling
temperature agarose (Biorad)
and pUC8 and pUC9 subolones of the XOII.S-Bgl
olones described by Boeshore ei_ a_l.23 were nick-translated and hybridized
to Southern transfers^ O r sequenoed by the Maxam-Gilbert method211.
31 nuclease protection and primer extension analysis
SI nuclease protection was carried out as described elsewhere19.
Annealing temperature was 50°.
For primer extension25, 500 ug of 3704 total leaf RNA was annealed to
the probe.
After 16 hours, the nucleic acids were preoipitated and then
redissolved in 50 mM Tris pH 8.3, 8 mM DTT, 10 mM MgCl2, 70 mH KC1, and 0.6
wU each of dATP, dTTP, dGTP, dCTP and 2800 u/ml reverse transcriptase (Life
Sciences, Inc.).
The primer was extended for 2 hours at 42°, RNa3e-
digested (Worthington/Cooper), SDS-phenol extracted and ethanol
precipitated.
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PETUNIA
ATGTTAGAAGGTGCAAAATCAATGGGTGCAGGAGCTGCTACAATTGCTTCAGCGGGAGCTGCTATCGG
NEUROSPORA
TC6T TTCC A6TCCSTTAS
*
*
ATGTTGCATAG
B
A
B
C
YSSE1AQ,
MQLVL
Fig. 1. Identification of the proteollpld gene. (A) Comparison of the £.
hybrlda mltoohondrial sequence and the N. orassa mltochondrial proteolipidlike gene. (B) Comparison of protein sequences of A: Putative P. hybrida
mltochondrial protein, B: Bovine nuclear-encoded protein, C: Putative ]J.
crassa mitochondrial protein, D: N. orassa nuclear-encoded protein, E:
yeast mitoohondrial-encoded proteTn. Identical amino acids are shaded.
RESULTS AHD DISCUSSION
Identification of the Petunia ATP synthase proteolipid subunlt gene
The Petunia proteolipid gene was identified when a region of P.
hybrlda 3701 mtDNA originally cloned in XO44.5 Bgl-1 was sequenced.
restriction map of this done has been described previously23.
A
We
originally identified our sequence as an atp 9 gene by searching the
organelle DNA library and protein sequence library of Genbank.
Comparisons
of the £. hybrlda coding region and derived protein sequence to the N.
crassa, bovine, and yeast sequences are shown in Figure 1.
The nucleic
acid sequence of the coding region of the Petunia gene is 56J homologous to
the N. orassa mltochondrial proteolipid-like gene (Fig. 1A) and 93>
homologous to the maize atp 9 gene^.
The amino acid sequence is identical
to that of the tobacco gene2& and specifies three additional amino acids at
the C-tenninus in comparison to the maize gene3.
The Petunia proteolipid
subunit gene (atp 9) codes for a 77 amino acid polypeptlde with two
hydrophoblc domains and a glutamic acid in the appropriate position to
allow for proton translocation and DCCD-blnding2?.
Of the 77 amino acids,
42 ar.e identical to the bovine subunit, 42 to the putative N. crassa
subunit, 38 to the nuclear encoded N. crassa subunit, and 34 to the yeast
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gCA6AT6AnCATACTA6CTT66TTAffiT(flWATnGTACWCCmnAm6AI6TMTATA6TATA*AC6CIG
*TT L£V fLO fLY ALA LYS U K
KIT H T
ALA
TCGTGAIGGAMMSCGTGM£A6MITCSM«TCtAT AT6 TTA CM (6T GCA MA TCA AIG ESI GCA
SLY ALA ALA THR 1LI
ALA Sill ALA H Y
*4*
* i * III
AY
ILt
CLY AM VAL L l l
SCR SCR
s u GCT GCT ACA AIT i n TIA GCC GGA fir GCT ATC M I Aft GCA we GTC en ACT Tec
UR
ILE HIS SCR VAL ALA A M
AJM P«t StH LID ALA LYS CLH L10 I'M
SLY TYR ALA
1L|
ICC ATI U T ICC C I * CCG CM M I CCA I U TIC 6 U M i CM TTA Tlf 661 TAT CCC ATI
LER U T M
H I LC* m
SLD ALA ILC ALA SIR m
ALA MQ HIT HT ALA PHI LEU UC
no cec m cn CIA ACC GM c a AII GCA ice TTT e a CCXAI6 AT6 6CC H I fie AIC
MR
PWC VAL m
C4.H VAL
A*f
1CA TIC CIA n c CM CIC CGI T * TUTCGmAMTGtSTGGGTMGUGGAGGGSA
Pit I
Xkol
EcoRI
••• HI
Fig. 2. Sequence of the Petunia hybrlda gene for the ATP aynthaae
proteollpld subunlt. Codon translation shown was according to the
universal genetic code. The arrows indicate the approximate 5' termini of
the 3 RNAs (see Fig. H) and the putative transcription start signal and
putative processing consensus sequences are underlined. The restriction
map of the area sequenced and the sequencing strategy is shown at the
bottom of the figure.
subunit (Fig. IB). Like other proteollpid subunits, the Petunia protein
does not contain tryptophan, but it is exceptional in that it contains a
hiatidine unlike the mammalian and fungal versions of this protein^.
DMA sequence of the P. hybrlda gene
The restriction map and nucleotide sequence of the Petunia atp 9 gene
and its 51 flanking region is shown in Figure 2.
nucleotides in length.
The coding region is 231
Almost all codon options are utilized.
However,
68J of the codons end in A or T.
Four upstream ATGs are present in frame with each other but not with
the coding region.
A sequence essential for mitochondrial ribosome binding
has not been established.
However, Dawson e^ al.-^ have proposed that plant
mitochondrial genes may carry signals analogous to the E. coll ShineDalgarno sequence.
At -10 to -17 in the Petunia gene is the sequence
AGAATTCG, which complements 6 of the 8 bases at the 3' end of the 18S maize
mt rRHA (TCCTAAGT).
Southern analysis of Petunia mltoohondrlal genomes with ATP synthase
proteollpld gene
Hybridization of mtDNAs of various Petunia genotypes (£.
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As
B
2
3
4
*
•
S
S
5
:
s
It
11
12
1.7 - *
- -
- «
6.0
4.2
35
Fig. 3. Southern analysis of ATP synthase sequences In Petunia lines. (A)
Restriction map of mitochondrial DNA around ATP synthase subunlt coding
region (heavy bar). Arrows Indicate the 5' terminus of the largest
transcript and the 3f end of the transcripts. (B) Total leaf DNA of
Petunia line 3704 (04), line 3688 (88) and line 3699 (99) was digested with
BamHI prior to electrophoresis and transfer to nitrocellulose. Lanes 1-3
were hybridized with a 5' flanking region probe of 2.9 kb (BamHI-Pstl).
Lanes 1-6 were hybridized with a 5' ooding region probe of 90 bp (TaqlTaql). Lanes 7-9 were hybridized with a 3' coding region probe of 180 bp
(Taql-BamHI), and lanes 10-12 were probed with a 3' flanking region
sequenoe of 1.1 kb (Bgll-BamHI).
hybrlda and CMS cytoplasms) with fragments of the atp 9 coding region
representing the 5' (lanes 1-6) and 3' portions (lanes 7-9) indicates that
more than one copy of homologous sequences exists in all 3 genomes.
Two
fragments in lines 3701 and 3688 and one fragment in 3699 hybridize to both
coding region probes (Fig. 3, lanes 1,5,7,8), while 3688 carries an
additional fragment (6.0 kb) hybridizing only to the 5' flanking and coding
region probe (Fig. 3, lane 5).
The gene we describe here is carried on the
3.5 kb BamHI fragment in line 3704.
A probe immediately 5' to the
transcribed region of the sequenced 3704 gene hybridizes only to the 3.5 kb
BamHI fragment in lines 3704 and 3699 (Fig. 3, lanes 1 , 2 ) . In 3688, the
same probe hybridizes to the 3.5 kb fragment and to the 6.0 kb BamHI
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1 2
3
4
5
6
—
—750
Fig. 4. SI nuoleaae analysis of transcripts of the Petunia atp 9 gene. SI
nuolease proteotlon by P. hybrida 3701 RNA of probe 5' end-labelled at the
BamHI site (see Fig. 2) and second cut at the Xhol site (see Fig. 2).
Arrows indicate the various RNA species' lengths. Lane 1: Haelll pBR322
molecular weight markers; lane 2: protection with total anther RNA; lane 3:
protection with total leaf RNA; lane 1: Alul pBR322 molecular weight
markers, 910, 659, 655, 521, and 103 bp; lane 5: protection with total
anther RNA; lane 6: protection with total ovary RNA; lane 7: protection
with mitochondrial RNA from suspension cells.
fragment which carries an amino-terminal segment of the coding region (Fig.
3, lanes 3, 5 ) . A probe immediately 3' to the transcribed region of the
sequenced 3704 gene hybridizes to only one 1.7 kb fragment in all 3 genome3
(Fig. 3, lanes 10-12).
Thus, while there is an additional region in 3701
and 3688 homologous to both portions of the coding region of the sequenced
3704 gene, only one complete gene with flanking regions homologous to the
sequenoed gene is present in all 3 genomes.
Restriction mapping,
hybridizations, and SI analyses show that the corresponding atp 9 gene in
the 3688 CMS genome is almost identioal to the 3701 £. hybrida gene that we
are reporting here (data not shown).
SI nucleaae and primer extension analysis of transcripts
The gene reported here diverges from the other homologous sequences
downstream of the stop oodon (data not shown).
Therefore a probe end-
labelled at the BamHI site immediately outside the coding region will not
be protected from S1 nuclease digestion by transcripts from any other
homologous gene.
Total RNA prepared from P. hybrida leaves protects 3
different DNA probe lengths, all of which extend through the coding region
(Fig. H, lane 3). The 5' end of the most abundant S1-resistant signal maps
approximately 121 bp upstream of the AUG start codon.
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Two additional minor
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signals map to positions 266 bp and 529 bp upstream of the AUG (positions
indicated in Fig. 2 ) . Shorter bands are likely due to RNA breakdown as
their location and quantity varies with different RNA preparations and time
in storage.
There ia only one 3' terminus at approximately 150 bp
downstream of the stop codon (data not shown).
The RNA species that
protect the DNA probe from SI digestion are found in the poly A minus
fraction when total leaf RNA is purified over oligo-(dT)-cellulose (data
not shown).
The ratio of abundance of each size class of RNA is approximately
1:5:100 as determined by counting gel slices in a liquid scintillation
counter and by comparison to quantity markers on autoradiograms (data not
shown).
There is no significant tissue-specific change in either the
lengths of the transcripts or their steady state ratios.
This ratio is not
detectably different in total RNA preparations from leaf (Fig. 4, lane 3)
nor anther tissues (lanes 2,5) nor in mitochondrial RNA preparations from
suspension cultures (lane 7 ) . The 3mall overall increase in protection by
ovary RNA (lane 6) is probably due to a higher proportion of mtRNA to total
RNA, since thi3 phenomenon is seen with other genes analyzed^.
To distinguish whether the discontinuities detected by nuclease S1mapping represented RNA splicing events or transcript termini, total leaf
RNA was analyzed by both S1 protection and primer extension.
A DNA primer
fragment was 5'-end labelled at the BamH1 site and extended by reverse
transcriptase from the EcoRI site.
S1 protection was performed in parallel
with a probe 5-end labelled at the same BamH1 site and extending out to the
Xhol site.
All three signals generated by S1 analysis are identical in
length as those obtained by primer extension (Fig. 5A) indicating that
these termini are not due to removal of introns but are actual transcript
ends.
No primer extension signals were seen corresponding to termini
beyond the Xhol site.
To pinpoint the exact origin of the longest transcript we have used
fragments 5 1 end-labelled at an Avail site found between the two longest
transcript termini.
The primer extension and S1 protection products of
this transcript were displayed next to a sequence ladder of this region
(Fig. 5B). Both methods show a transcript origin Just following the
sequence ATATAGTA.
This represents the best match of a plant mitochondrial
5' termini sequence to the yeast mitochondrial promoter sequence yet found
(Table 1.A). The yeast atp 9 (oli1) gene begins transcription at the last
nuoleotide of the sequence ATATAAGTA10.
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B
1 2
3
PS
G A T C
Probe
Figure 5. Primer extension analysis of Petunia atp 9 gene transorlpts.
(A) Lane 1~ Primer extension of an EcoRl-BamHI probe (see Fig. 3A; 5'
end-labelled at the BamHI site after annealing with total leaf RNA. Lane
2: SI nuclease protection of Xhol - BamHI probe. Lane 3: Alul pBR322
molecular weight markers. (B) Primer extension of longest RNA shown next
to a sequence ladder. Probes were end-labelled at the Avail site at 389. P: Prlaer-extended produot; S: S1-protected product; G,A,T,C:
sequence lanes. Sequence ladders of the non-coding strand have been
transposed for ease of reading.
In the maize oox I gene, the sequence TCATAAGTA, whioh has seven out
of nine homology to the yeast promoter sequence, was found where the
longest of the two detected transcripts mapped".
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Table 1.
A.
Sequence homologies In plant mitochondrial
genes 5' to protein coding regions
Sequence homology to the region preceding tha
5' terminus of the longest Petunia atp 9 transcript
A T A T A A G T A
Yeast Initiation
atp 9
B.
I IIII
"° A T A T A
Sequences homologous to the region at the 5' terminus
of the Intermediate-size Petunia atp 9 transcript
Petunia atp 9
"256A
Pea cox II
Oenothera cox II
Maize cox I
I I
I I I I I I I
A A A T T T A C T'A
A G A G A A G A
-q,l I I I I I
. I I I I I I I I
" A
A A T C A C G T ' A A G T G A A G A
I I
I
I I I I I I ITI
o
21
A A A T C T C G TTA T G A G A A T C
III
II
I I I I I
II
-165
"A A A
C T C A T*A A G T
A A T
1 1
1 1
1 1 1 1 1
1 1 1
A A A T T T C N T*A A G
Consensus
Wheat cox II
Rice cox II
Oenothera cox II
A
GAA
T
-97AI IA IA IG T IT CI G T IA IA I AI GI AI A IT G
I I I I
A A A T
II
I
-82A A
C
-252
G
l"l I
I I
T A A A A G
I I I
I I
A A T A A A A G
I
A
I
A
I
A G A
I G
A G A
Sequences homologous to tha region surrounding the
5' terminus of the shortest Petunia atp 9 transcript
Petunia atp 9
Maize cox I
Wheat cox II
V
I
*
A A T T T C A T ^ A A G A T A A G
-311 I I I
Pea cox II
C.
IIL
G T A*
-124,,
CCT
T
A T G C T T T G
I II
I I IIII I
66
C CTTT C A T T C T T T G
I III
I III I
-60
ecu
T C C T T T C
denotes mapped termini
denotes homology (In B, homology to consensus)
method reported approximated BNA terminus
nuclease mapped termini from the Petunia atp 9 and other plant
mitochondrial genes we have discovered an extended sequence homologous to
the maize TCATAAGTA region (Table 1.B).
(AAATTTCATAAGATAAGA)
Since this extended sequence
is found in the regions flanking the terminus of the
Intermediate-size Petunia transcript, we propose that the maize sequence
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may signal transcript processing rather than initiation.
The end of the
RNA map3 internal to this sequence, unlike the longest Petunia atp 9
transcripts, which map Just downstream of the putative promoter.
While the
maize oox I terminus does have homology to the entire extended sequence,
the putative Petunia atp 9 promoter does not.
Furthermore, there may be
other maize cox I transcripts which were not detected because of low
abundance and/or RNAs longer than the probe used.
Therefore, insufficient
data exists to conclude whether the maize cox I sequence serves as a
promoter or as a processing signal.
The 5' termini of the two mapped pea cox II transcripts are also
homologous to the terminus of the Petunia intermediate-size transcript
(Table 1.B). The two pea transcripts map at -302 and -285 relative to the
start codon and are present in a 1:5 ratio.
Because Moon e_t al^.12 used a
probe spanning -363 to +56 of the pea gene, any longer transcript analogous
to the Petunia primary transcript would not have been detected.
For
neither the maize nor pea genes was a primer extension performed which
would have detected transcripts longer than the probe.
Using the data from maize, pea, and Oenothera, Hiesel and Brennicke2"
suggested a consensus sequence AAATYNNNTAAG for 5' termini of mitoohondial
RNAs.
Adding data from Petunia wheat, and rice, we propose the extended
consensus AAATTTCNTAAG*GAA (Table 1.B). Although we have found regions of
homology in the wheat cox II" and rice cox II1? genes, the transcript
termini for these genes have not been reported so it remains to be
determined if they do, in fact, terminate at the sites predicted.
The
homologous -82 region in Oenothera (Table 1.B) was not found to be a site
of transcript termination28.
The -82 sequence in Oenothera may thu3
provide a clue to the constraints on this signal.
At the region where the third and shortest Petunia transcript maps is
the sequence CCTATGCTTTG which has 10 bases in common with the sequence
CCTTCATTCTTTG located at the terminus of the shorter of the two detected
maize sequences (Table 1.C). No similar sequence is present between the
two map points of the pea cox II transoripts and the pea gene's ATG codon.
However, there is a homologous region at -60 in the wheat cox II gene.
S1 mapping of the transcripts terminating at the sequence shown in
Table 1, sections B and C, show that the ends are internal to these
consensus sequences.
The majority of these RNA termini do not have
sequences resembling promoter elements, and the two shorter Petunia RHAa
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are definitely not due to splicing.
Therefore, we propose that plant
mitochondrial promoters resemble the yeast promoter ATATAAGTA, and that the
two other consensus sequences are processing signals.
Since many yeast
mtRNAs are processed by a clipping mechanism, it may be that this is also
the method used in plant mitochondria.
No dicot mitochondrial gene has
been found containing an intron; however, few dlcot mitochondrial genes
have yet been sequenced.
Because certain monocot gene3 (rice cox II 2 9,
wheat cox 11°, and maize cox II") do contain introns, perhaps more than one
form of processing is used within the same transcript.
CONCLUSION
We report the sequence of the Petunia atp 9 gene and its transcript
structures.
We have also determined consensus sequences/signals that are
probably involved in the generation of these transcripts.
The three
transcripts mapped in Petunia and those mapped in maize, Oenothera and pea
could be due formally either to transcription initiation or processing.
However, the excellent match of the Petunia -536 sequence to the yeast
mitochondrial promoter element makes initiation the most likely source of
this transcript terminus.
The conserved sequences at the other 5' termini
in the Petunia, pea, and maize genes could be alternative promoters or
processing signals.
5* transcript processing has recently been established
in another plant organelle.
The
maize chloroplast rboL gene's largest
transcript initiates at -300, and the presence of the -105 and -63
transcripts were shown In vitro to depend on transcription Initiation at
the -300 promoter30.
xhe -63 transcript could be produced from a -300
transorlpt by a chloroplast extract^O.
Absolute determination of the
origin of plant mitochondrial transcripts must await the development of ^n
vitro transcription and processing systems or mitochondrial transformation.
ACKNOWLEDGEMENTS
This work wa3 supported by the U.S.-Israel Binational Agricultural
Research and Development Fund and NSF grant PCM-8101281. We thank Shamay
Izhar for Petunia lines, Haury Boeshore for the original lambda clones, and
Bill Pearson and the University of Virginia Academic Computing Center for
SEQHENU.
•To whom correspondence should be addressed
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