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J Biol Chem, Vol. 273, Issue 45, 30003-30011, November 6, 1998
From the A relatively thermostable 22-kDa endoribonuclease
(MAR1) was purified more than 10,000-fold from a mitochondrial extract
of Leishmania tarentolae and the gene cloned. The purified
nuclease has a Km of 100-145 ± 33 nM and a Vmax of 1.8-2.9 ± 2 nmol/min, depending on the RNA substrate, and yields a 3'-OH and a
5'-phosphate. Cleavage was limited to several specific sites in the
substrate RNAs tested, but cleavage of pre-edited RNAs was generally
independent of the addition of cognate guide RNA. The MAR1
gene was expressed in Escherichia coli or in L. tarentolae cells, and the recombinant protein was
affinity-purified. The cleavage specificity of the recombinant enzyme
from L. tarentolae was identical to that of the native
enzyme. The single copy MAR1 gene maps to an 820-kilobase
pair chromosome and contains an open reading frame of 579 nucleotides.
The 18-amino acid N-terminal sequence shows characteristics of an
uncleaved mitochondrial targeting sequence. Data base searching
revealed two homologues of MAR1 corresponding to unidentified open
reading frames in Caenorhabditis elegans
(GenBankTM accession number Z69637) and
Archaeoglobus fulgidus (GenBankTM accession
number AE000943). The function of MAR1 in mitochondrial RNA metabolism
in L. tarentolae remains to be determined.
The processing of mitochondrial RNAs shows great variation between
species. In mammalian mitochondria, both DNA strands are completely
transcribed, and the transcripts are processed by the excision of
interspersed tRNAs (1, 2). In Saccharomyces cerevisiae
mitochondria, 5' end maturation of tRNAs is carried out by a
mitochondrial RNase P (3), and RNA degradation may involve a 3'-5'
exonuclease activity (4). The 3' end processing of the cytochrome
b (Cyb)1 mRNA
in yeast is mediated by the product of the nuclear gene CBT1
(5). In plant mitochondria, inverted repeats at the 3' ends of
protein-coding genes serve as processing signals for 3' end maturation
(6), and RNase Z has been identified as the nuclease responsible for
the specific processing of the 3' ends of tRNAs (7).
In the kinetoplast mitochondrion of the trypanosomatid protozoa,
Leishmania tarentolae and Trypanosoma brucei, the
maxicircle DNA encodes two rRNA genes and 18 potential protein-coding
genes, 12 of which are cryptogenes whose transcripts are edited by the insertion and deletion of uridine residues usually within coding regions (8-11). RNA editing reactions appear to be initiated by specific cleavages of the pre-edited mRNAs, mediated by base
pairing with specific cognate guide RNAs (gRNAs) (12, 13). Little is
known about the processing and turnover of mitochondrial RNAs in these
cells, either in terms of specific cis-acting signals or enzymatic
activities. Three different endoribonuclease activities, separable by
sedimentation or anion exchange chromatography, have been identified in
a mitochondrial extract from T. brucei (14). One of these
activities, which sedimented at 20 S in glycerol gradients, exhibited a
gRNA-dependent cleavage at the first mismatch upstream of a
duplex RNA region (14-16), precisely as predicted by the enzyme
cascade model for RNA editing (12). Another activity, which sedimented
at 15 S and was independent of added gRNA for cleavage, might
correspond to an endoribonuclease activity that has been described
previously in crude mitochondrial extracts from both L. tarentolae and T. brucei (17, 18). The endoribonuclease activities in the crude extracts were both shown to cleave pre-edited Cyb mRNAs two nucleotides upstream of the first editing site. However, the T. brucei activity had specificity for the
pre-edited Cyb sequence and did not to cleave the mature edited sequence.
In addition to a gRNA-dependent editing endoribonuclease,
additional nucleases are presumably necessary for kinetoplastid mitochondrial mRNA maturation. There is some evidence for
polycistronic transcription of the maxicircle (16, 19-22). Primary
transcripts are then subjected to 5' processing and 3' end cleavage,
followed by 3' polyadenylation and polyuridylylation. In addition, many of the mitochondrial protein-coding genes have overlapping 5' and 3'
ends, and the maturation of such transcripts could represent an
additional level of gene regulation (19). Clearly, multiple specific
ribonucleases must be required for the processing and turnover of
rRNAs, mRNAs, gRNAs, and tRNAs in the mitochondria of these
organisms. However, to date, the only ribonuclease purified to
homogeneity from kinetoplastid mitochondria is RNase H (23, 24).
In this paper we describe the isolation and characterization of MAR1
(for Mitochondrial Associated
Ribonuclease) from a mitochondrial extract of L. tarentolae. MAR1 was purified to homogeneity and the
MAR1 gene cloned and expressed. Further biochemical
characterization and genetic analysis should help elucidate the role of
this nuclease in the processing of RNAs within the mitochondrion of
Leishmania.
Strains and Culture Conditions--
L. tarentolae UC
strain cells were grown in brain heart infusion (Difco) supplemented
with hemin (1.6 µM) (Sigma). For small-scale mitochondrial preparations (1-2 liters of culture or less), cells were
grown with rotation in a Cell Production Roller Apparatus (Bellco) at
27 °C. For large-scale mitochondrial preparations (15 liters), cells
were grown in a BioFlo IV fermentor (New Brunswick Scientific). The
cells were harvested with a Masterflex tangential filter apparatus
(Millipore). For purification of mitochondria, cells (1.5-1.8 × 108 cells/ml) were harvested by centrifugation at 5000 × g for 10 min at 4 °C. Cells were lysed in hypotonic
Tris-EDTA, and the mitochondria were purified by isopycnic flotation in
Renografin gradients as described previously (25). The mitochondrial
fractions had less than 5% cytosolic contamination. Mitochondria were
stored at Extract Preparation--
Renografin-purified mitochondria (1.0 g/ml, wet weight) were thawed on ice and solubilized by adding MSB
buffer containing CHAPS and ammonium acetate (10 and 500 mM
final concentration, respectively). A protease inhibitor mixture (0.5 µg/ml leupeptin, 1 µg/ml pepstatin, 0.01 µg/ml chymostatin, 0.1 mM benzamidine, 0.1 mM phenylmethylsulfonyl
fluoride) was also added to the mitochondrial extract, which was kept
at 4 °C for 30 min and then clarified at 12,000 × g
for 20 min. The extract was then heated at 55 °C for 20 min. This
heating step causes a large percentage of the proteins to coagulate
while MAR1 remains in solution. The heated extract was further
clarified by centrifugation at 100,000 × g in a
Beckman TLA-100.4 fixed angle rotor for 1 h in a Beckman Optima
tabletop centrifuge. The supernatant (S-100) was saved and the pellet
discarded. The S-100 (100 ml) was dialyzed against 4 liters of MSB
buffer and either stored at MAR1 Purification--
The S-100 supernatant (in MSB buffer) was
loaded onto a Mono Q HR5/5 (anion exchange) column (Amersham Pharmacia
Biotech) at a flow rate of 0.5 ml/min at 4 °C. After loading, the
column was extensively washed with 30 ml of MSB containing 1 mM CHAPS (QA buffer) until no more detectable protein
eluted. The column was developed with a linear gradient of 20 ml of QB
buffer (QA buffer containing 1 M KCl). Individual fractions
(0.5 ml) were assayed for endoribonuclease activity. Fractions
containing the peak of nuclease activity were pooled, concentrated with
an Ultra-Free 15 concentrator (Millipore), and loaded onto a Superose
12 column (Amersham Pharmacia Biotech) (equilibrated with 0.1 M triethanolamine (pH 8.0)). The Superose 12 column was
developed with 30 ml of 0.1 M triethanolamine (pH 8.0) at a
flow rate of 0.3 ml/min. Fractions containing the peak of nuclease
activity were pooled and loaded onto a Mono P HR5/5 column (Amersham
Pharmacia Biotech) at a flow rate of 0.5 ml/min at 25 °C. Proteins
bound to the Mono P column were eluted with 20 ml of Polybuffer 74 (Amersham Pharmacia Biotech) (pH 5.0), allowing a pH gradient to form
in the column. Individual fractions (0.5 ml) were assayed for
endoribonuclease activity and peak fractions pooled and concentrated
with an Ultra-Free 15 concentrator. The Mono P-pure MAR1 was
gravity-loaded onto a PD-10 column (Amersham Pharmacia Biotech)
equilibrated with MSB buffer and then eluted in MSB buffer containing
10% glycerol and stored at In Vitro Transcription and Labeling of Synthetic RNA--
Cyb
(fully edited and pre-edited) and ND7 (pre-edited) mRNAs were
transcribed using T7 RNA polymerase and plasmids containing the
templates for the various transcripts under the control of a T7
promoter. Plasmids were linearized by restriction digestion, the
linearized plasmids were incubated in an in vitro
transcription mixture containing T7 RNA polymerase, nucleotides, and
the appropriate buffer (24). The resulting synthetic RNAs (CybNE,
CybFE, and ND7) were 3' end-labeled by ligation of
[ Enzyme Assays--
Either 5' or 3'-labeled RNAs were incubated
at 27 °C for 30 min in 50 mM HEPES (pH 7.5), 1 mM DTT, 20 mM KCl, 10 mM
MgCl2, 0.2 mM EDTA buffer (HKM buffer), in the
presence or absence of enzyme. Samples (100 µl) were extracted with
100 µl of Tris/EDTA-saturated phenol/chloroform (pH 8.0). The RNA in
the aqueous phase was precipitated with 1/10 sample volume (10 µl) of
3 M sodium acetate (pH 5.2) and 3 volumes (300 µl) of
ethanol, with 1 µg of glycogen as carrier. Samples were pelleted by
centrifugation in an Eppendorf microcentrifuge at 14,000 × g at 4 °C for 30 min. The pellets were washed with 1 ml
of 70% ethanol, dried under vacuum, and resuspended in denaturing buffer (8 M urea/0.5% SDS, 0.3% xylene cyanol, 0.3%
bromphenol blue, 2 mM EDTA, 10 mM Tris-HCl (pH
8)). The samples were heated at 95 °C for 5 min and
electrophoretically separated in 7 M urea/6% (or 10%)
acrylamide at 500 V for 2 h. Gels were transferred to 3MM blotting
paper (Whatman), dried under vacuum at 85 °C for 45 min, and then
exposed to a PhosphorImager screen (Molecular Dynamics). For analysis
of the initial velocity kinetic constants, increasing concentrations of
5'-labeled synthetic mRNA substrates were incubated as above with
constant concentration of enzyme (10 ng). Reactions were carried out at
27 °C for 30 min, and samples were processed as above. The velocity
of conversion of full-length mRNA into cleaved products was
calculated and plotted as a function of substrate concentration. These
data were plotted as double-reciprocal plots, allowing for calculation
of the first order rate constants (Km and
Vmax) for the various substrates.
Oligodeoxynucleotides--
Oligodeoxynucleotide primers for PCR
amplification, RT-PCR, hybridization, and primer extension assays were
synthesized by standard phosphoramidite methods (Life Technologies,
Inc.) and purified by electrophoresis in an 8 M urea/8%
polyacrylamide gel at 250 V. The band was visualized by UV (256 nm)
shadowing on a 20 × 20 k6F Silica Gel Thin Layer Chromatography
Plate (Whatman) and eluted from the gel matrix in 0.3 M
sodium acetate at 55 °C, 30 min, followed by ethanol precipitation
and resuspension in water. The following oligonucleotides were utilized
in this study: S-2255,
5'-CTGIAGTTC(C/I)ACGCC(C/I)AGGAA(C/I)GC(C/I)GTCTT-3'; S-2271,
5'-TTGAATTCGCATTGAGCACCTGCTTTTTTTTTTTTTTTT-3'; S-2272, 5'-TTGAATTCGCATTGAGCACCTGC-3'; S-2273,
5'-AACTAACGCTATATAAGTATCAGTTTCTGTACTTTATTG-3'; S-2323,
5'-CGG(T/C)CGCGTGAC(T/C)TCCAGGTG-3'; S-2356,
5'-ACTACCGGGAAAATTGTGTCG-3'; S-2357, 5'-CTGGAGTTCCACGCCGAGGAAG-3';
S-2473, 5'-CTTGGAGATCTACGCACAGAAACGCTG-3'; S-2545,
5'-CTGCAGTCTCTAGACTTCACTTGTCATCGTCATCCTTGTAGTCAAGCTT-3'; S-2546,
5'-GCTAGGCATGGATCCATGCCTCGCTTGATGCCGCATTATTCTACG-3'; S-2630, 5'-GATTTCGAAGCTACTGAGGGAGG-3'. The nucleotides in parentheses represent degenerate positions and (I) represents inosine.
Peptide Isolation and N-terminal Sequencing--
The purified
protein was electrophoretically separated in a 10%
acrylamide/Tricine/SDS gel (Bio-Rad). The purified protein was
electroblotted onto a polyvinylidene difluoride membrane and visualized
by staining for 1 min with 0.1% Coomassie Blue R-250 in 50% methanol,
followed by 2-min destaining with 50% methanol. The portion of the
membrane containing the stained band was excised and subjected to
N-terminal sequencing on an Applied Biosystem 477A automated sequencer.
The following sequence was obtained: MPRLMPHYSTSKTAFLGVDLQCAG.
Nested RT-PCR Reactions--
Based on the 24 N-terminal amino
acids sequence obtained from the purified MAR1 protein, the degenerate
oligonucleotides S-2323 (GACQLDV) and S-2255 (QLDVGLFATK), which were
complementary to MAR1 mRNA, were used in a nested RT-PCR
reaction to obtain the 5' sequence. S-2323 was used as primer for the
synthesis of the first-strand cDNA in a reaction containing 1 µg
of poly(A)+ RNA from L. tarentolae; 120 pmol of
S-2323 and 200 units of Superscript RT-II (Life Technologies, Inc.) in
a 20-µl reaction volume, according to the manufacturer's
instructions. A 2-µl aliquot of the cDNA reaction was used as
template in a PCR reaction containing 100 nM S-2323 and the
L. tarentolae spliced leader RNA (slRNA)-specific primer,
S-2273. Amplification of the MAR1 5' sequence was performed in a GeneAmp 9000 thermocycler (Perkin-Elmer) in a reaction mixture containing 20 mM Tris-HCl (pH 8.0), 50 mM KCl,
1.5 mM MgCl2, 200 mM dNTPs, and 5 units of Taq polymerase, in a final volume of 100 µl. The
sample was denatured for 2 min at 94 °C, followed by 30 cycles of
denaturation at 94 °C for 0.5 min, annealing at 40 °C for 0.5 min, and extension at 72 °C for 1 min. An aliquot of the first PCR
reaction (5 µl) was used as a template for a second PCR reaction
containing the degenerate primer S-2255 and the slRNA-specific primer,
S-2273, as described above. The PCR products were separated in a 2%
agarose-TAE (0.04 M Tris acetate, 0.001 M EDTA
(pH 8.0)) gel containing 0.5 µg/ml ethidium bromide and visualized by
UV light. A 130-bp DNA fragment was cloned into the pGEM-T Easy vector
(Promega), according to the manufacturer's instructions, and
transformed into Escherichia coli DH5 DNA Ligation and Colony Hybridization--
DNA ligations were
performed as described (Invitrogen) using the pCR 2.1-TOPO cloning kit
(Invitrogen). Colony hybridization experiments were performed as
described (26).
Northern Analysis--
A Northern blot of 6 µg of total RNA
and 2 µg of poly(A)+ RNA isolated from L. tarentolae cells was performed in a 18% formaldehyde/1.5% agarose gel (16 h, 1.7 V/cm). The RNA was transferred to a Zeta-Probe membrane (Bio-Rad), which was hybridized with either end-labeled oligonucleotide primers or the random-primed MAR1 probe
(Fig. 7A) (Prime-IT II kit, Stratagene).
DNA Sequencing and Generation of Nested Deletions--
DNA
sequencing of cloned inserts was performed manually by the
dideoxy-termination reaction with 10 pmol of plasmid DNA template and
Sequenase V.2.0 (Amersham Pharmacia Biotech), according to the
manufacturer's instructions. The sequencing products were then
separated in an 8 M urea/6% polyacrylamide gel and
visualized by autoradiography. The L. tarentolae 3 kilobase
pair genomic fragment containing the MAR1 gene sequence was
cloned into the EcoRI-BamHI sites of the
pBluescript II SK(+) vector (Stratagene), generating pB1-MAR1. The
pB1-MAR1 plasmid was linearized by digestion with KpnI and
EcoRI, to generate an Exo-III-resistant restriction site and
an Exo-III-sensitive site. Ten 400-bp nested deletion fragments were
generated with the Erase-A-Base kit (Promega), according to the
manufacturer's instructions. Five clones from each deletion time point
were sequenced by the Applied Biosystems-Perkin-Elmer automated
sequencer (Applied Biosystems-Perkin-Elmer). This approach ensured the
sequencing of each nucleotide from at least three independent clones in
both directions.
Contour-clamped Homogeneous Field Electrophoresis
(CHEF)--
L. tarentolae genomic DNA blocks
were prepared according to Van der Ploeg (27) and separated in 1%
agarose-1/2 TBE in a CHEF apparatus (28) at 200 V for 15 h with
1-min pulses, followed by electrophoresis for 9 h using 90-s
pulses. The chromosomal bands were visualized by ethidium bromide
staining and the gel photographed using a C-80 Epi-illumination UV
Darkroom (Ultraviolet Products). The DNA was transferred to a
Zeta-Probe membrane (Bio-Rad), according to the manufacturer's
instructions and hybridized to the MAR1 gene random-labeled
using the Prime-IT kit (Stratagene).
Mapping the 5' and 3' Terminus of the Mature MAR1
mRNA--
The 5' terminus of the MAR1 mRNA was defined by
RT-PCR in a reaction containing the oligonucleotides S-2474, which is
antisense to the MAR1 mRNA, and S-2273, which
corresponds to a part of the spliced leader sequence. The PCR products
were separated in 2% agarose-TAE and cloned into pCR 2.1-TOPO
(Invitrogen), and transformed into competent E. coli DH5
The mapping of the 3'-untranslated sequence of the MAR1
transcript was performed by a modification of the procedure described above. cDNA synthesis from poly(A)+ RNA was performed
using an oligo(dT) primer, S-2271, and Superscript II RT (Life
Technologies, Inc.). The cDNA mixture was incubated for 30 min at
25 °C and then for 1 h at 45 °C. An aliquot of 5 µl of the
cDNA synthesis reaction was PCR-amplified with the primers, S-2272
and S-2356, in a standard PCR reaction mixture (Promega). The sample
was subjected to 2-min denaturation at 94 °C followed by 30 cycles
of denaturation at 94 °C for 0.5 min, annealing at 60 °C for 0.5 min, and extension at 72 °C for 2 min and a final step of 72 °C
for 10 min. A 5-µl aliquot of the first PCR reaction was subjected to
a second PCR amplification in a standard PCR reaction mixture (Promega)
with the primers S-2272 and S-2630, using the same conditions as
described for the first PCR amplification. The PCR products were
separated in 1% agarose/TAE gel and the amplified DNA was cloned into
the pCR 2.1-TOPO vector (Invitrogen).
RNA Mapping and Sequencing--
5'-Labeled in vitro
synthesized RNAs (Cyb or ND7) were incubated with sequence-specific
nucleases (T1, U2, Phy M, and Bacillus cereus), as described
in the RNA Sequencing Kit (Amersham Pharmacia Biotech). These reactions
were used as sequencing ladders in 10% acrylamide/7 M urea
gels together with MAR1 cleavage reactions with the same mRNAs. To
assay for gRNA dependence of MAR1 cleavage, reactions were performed in
the presence and absence of gRNAs and separated as above.
Sequence Analysis of MAR1--
The available sequence data bases
(GenBankTM nonredundant data base and Swissprot) were
searched using the predicted polypeptide deduced from the
MAR1 open reading frame. The search programs employed were
BLASTP (National Center for Biotechnology Information at
http://www.ncbi.nlm.nih.gov/index.html), using a Blosum62 data matrix,
and BLITZ (European Bioinformatics Institute at
http://www.ebi.ac.uk/searches/searches.html). The program CLUSTALX was
used to align the amino acid sequence of L. tarentolae MAR1,
C. elegans, Bacillus subtilis, and
Archaeoglobus fulgidus. Manual refinements were performed to
optimize the alignments, taking into consideration the predicted
secondary structure obtained from the individual polypeptides. Predict
Protein (PHDsec)
(http://www.ebi.ac.uk/searches/searches.html) was used to perform the
secondary structure predictions. The Monte Carlo algorithm implemented
by the RDF2 program (GCG package) on a VAX 4000 computer was used to
evaluate the statistical significance of the alignments. The result is
shown as S.D. values (standard deviations from the mean of randomized
sequences) of 200 rounds of randomization with a ktuple of 2. The
isoelectric point of MAR1 was determined with the programs ISOELECTRIC
(GCG package) and MultiIdent
(http://expasy.hcuge.ch/www/expasy-top.html). The N terminus
amphipathic nature of MAR1 and C. elegans sequences was
determined with the program HELICALWHEEL (GCG package).
Expression and Purification of the Recombinant MAR1 Protein and
Generation of Polyclonal Antiserum--
The MAR1 gene was
used to express a histidine-tagged (His-tagged) version of the MAR1
protein in E. coli cells or an epitope-tagged version in
L. tarentolae cells. For E. coli expression, the
coding sequence of the MAR1 gene was cloned into the pQE-31
expression vector (Qiagen), which places the gene under the control of
an isopropyl-
Recombinant MAR1 protein expressed in E. coli was used for
the production of polyclonal antiserum in rabbits (Animal Pharm Services, Inc.). A 1:10,000 dilution of serum, from the seventh cycle
of immunization, was used for Western analysis.
For expression in L. tarentolae, the MAR1 gene was ligated
into the pX vector (29). Prior to ligation, a PCR reaction was carried
out with oligonucleotides S-2545 and S-2546, which are complementary to
the 5' and 3' end, respectively, of the MAR1 coding region. These
oligonucleotides were designed to insert a BamHI site at the
5' end and an XbaI site at the 3' end of the MAR1 coding
sequence. In addition, the 3' oligomer, S-2545, carries the sequence
coding for the FLAG-epitope (N-Asp Tyr Lys Asp Asp Asp Asp Lys-C)
(Eastman Kodak Co.). The PCR product generated by amplification of MAR1
with oligonucleotides, S-2545 and S-2546, was digested with
BamHI and XbaI and cloned between the
BamHI and XbaI sites of pX, generating the
plasmid pXMARFLAG. The pXMARFLAG plasmid was used to transfect L. tarentolae cells. Transformants were selected by the
G418-resistant phenotype provided by the plasmid-derived marker (29).
Two liters of pXMARFLAG-transformed L. tarentolae cells were
grown in brain heart infusion medium containing 200 µg/ml of G418 for
14 h at 27 °C and harvested as described above. The pellet was
suspended in 50 ml of MSB and sonicated at medium power. The extract
was centrifuged at 100,000 × g and loaded onto a 1-ml
FLAG-affinity column (Kodak). The cleavage activity and specificity of
the recombinant MAR1 were determined as described above.
Optimum Temperature and Divalent Cation Requirements--
In crude
mitochondrial extracts the optimal temperature for the MAR1 reaction is
27 °C (data not shown). However, the activity was relatively
resistant to heating at 55 °C for 20 min, and the thermal stability
was independent of the presence of RNA substrate. As shown in Fig.
1A, 70% of the activity in
the crude extract remained in the supernatant after the 55 °C
heating step. A similar stability against thermal denaturation was
observed with purified MAR1 (data not shown), indicating that this is
an intrinsic characteristic of the enzyme and not due to some
extraneous factor.
MAR1 has an absolute requirement for divalent cations (Fig.
1B). When the chloride salts of Mg2+,
Mn2+, Zn2+, and Ca2+ were used in
the cleavage reaction, Mg2+ (up to 10 mM) was
the preferred cation, but Mn2+ also could satisfy this
requirement, while Zn2+ and Ca2+ worked to a
much lesser extent.
Purification of MAR1--
Due to the relative thermal stability of
MAR1, heating of a 12,000 × g for 20 min clarified
CHAPS lysate at 55 °C was used as the initial step in purification
(Table I), followed by centrifugation at
100,000 × g for 1 h. Chromatography of the
resulting S-100 supernatant through Superose 12, Mono Q (Fig.
2A), and Mono P (Fig.
2B) yielded a 16,000-fold enrichment of specific activity (Table I). Analysis of the peak fractions by SDS-acrylamide gel electrophoresis and silver staining is shown in Fig. 2C. The
Mono P peak showed a major 22-kDa band that represented 97% of the protein in this fraction. No changes in substrate cleavage patterns were observed between the enzyme activity in the crude extract or the
Mono P fraction (data not shown).
Kinetic Analysis of MAR1 and Cleavage Specificity--
To make an
initial analysis of the cleavage specificity of MAR1 and also to
examine the possibility that MAR1 is involved in RNA editing, cleavage
sites were mapped on two different pre-edited mRNA substrates (ND7
and Cyb) and on one fully edited mRNA substrate (Cyb), using the
biochemically purified enzyme and 5' or 3' end-labeled substrates. As
shown in Fig. 3A, two major
cleavage sites were located at editing site 2 (ES2) and editing site 3 (ES3) of pre-edited ND7 mRNA. Another major cleavage site was
located just upstream of editing site 7 (ES7), and five additional
cleavage sites were located downstream of the pre-edited region (PER).
The locations of the major cleavage sites provided no evidence for
sequence specificity, but it is also apparent that the digestion is not random. Digestion with a large excess of enzyme for extended periods led to complete cleavage of the substrate to short oligomers (8-10 nucleotides) (data not shown).
Also shown in Fig. 3A, the addition of excess cognate gRNA
for block I of ND7 did not induce a cleavage at ES1 as would be predicted for a nuclease involved in the initial step of gRNA-mediated editing. Instead, the cleavages at ES2 and ES3 were inhibited and the
major cleavage upstream of ES7 was unaffected, as were the downstream cleavages.
In the case of the pre-edited Cyb substrate, the precise cleavage sites
were not determined. However, major cleavages located at or near ES1
and at several sites within the 3' region of the RNA downstream of the
ER were observed (Fig. 3B). A 5' end-labeled substrate
yielded identical results as the 3' end-labeled substrate shown (data
not shown). Use of a fully edited Cyb substrate led to the
disappearance of one of the 3' cleavages, but there was no effect on
the ES1 cleavage. To test the possibility that there could still be a
substrate preference for pre-edited mRNAs, a kinetic analysis was
carried out under initial velocity conditions. With all of the
substrates tested, MAR1 cleavage activity followed Michaelis-Menten
kinetics with a Km of 120 ± 15 nM
and a Vmax of 2.8 ± 2 nmol/min for the
pre-edited Cyb mRNA (Fig.
4B) and a
Km of 100 ± 10 nM and
Vmax of 2.9 ± 0.5 nmol/min for the fully
edited version of the same mRNA (Fig. 4C). Similar values for Km and
Vmax were obtained (Fig. 4A) with the pre-edited ND7 substrate. We conclude that both pre-edited and fully
edited RNAs were cleaved by MAR1 with comparable efficiency.
Cloning and Sequencing of the MAR1 Gene--
A 24-amino acid
N-terminal sequence (MPRLMPHYSTSKTAFLGVDLQCAG) was obtained by
microsequence analysis of the purified MAR1 protein. Based on this
sequence, the degenerate S-2323 primer was used to synthesize the
first-strand cDNA. This cDNA was then subjected to RT-PCR
amplification utilizing the S-2323 3' primer and the slRNA-specific
S-2273 5' primer. The product of the first PCR reaction was
subsequently re-amplified in a nested PCR reaction with the degenerate
S-2255 3' primer and the S-2273 5' primer. A 130-bp DNA fragment was
cloned and sequenced. The sequence of the 130-bp fragment revealed
three amino acid differences
(MPRLMPHYSTSKTAFLCVDLQEAF) (shown in
boldface letters) from that of the N-terminal sequence obtained from
microsequencing. These amino acid differences are toward the C terminus
of the peptide and represented ambiguous amino acid assignments.
However, the internal deduced amino acid sequence upstream of primer
S-2255 (STSKTA) confirmed the identity of the cloned fragment (Fig.
5).
The 130-bp PCR product hybridized with a 3-kb
EcoRI-BamHI genomic fragment. The 3-kb region of
EcoRI-BamHI-digested genomic DNA was cloned into
the pGEM-7Zf(+) vector. The MAR1-containing plasmid was
identified by colony hybridization with the 5'-labeled MAR1-specific
S-2356 oligonucleotide and the sequence of the insert DNA determined.
This sequence contains a single open reading frame of 579 nucleotides
that encodes for a protein of 192 amino acids with a molecular mass of
21.6 kDa and a predicted isoelectric point of 7.38 (Fig. 5). RT-PCR of
the 5' region of the transcript indicated the presence of two
alternative splice acceptor sites for the trans-spliced 39-nt slRNA
(data not shown). One site (SAS-1) is located at position Recombinant MAR1 Shows Identical Specific Activity and Cleavage
Specificity as Native MAR1--
The MAR1 gene was expressed
in E. coli cells as a His-tagged protein and purified by
affinity chromatography. The specific activity of the E. coli-expressed MAR1 was 200-fold lower than that of the native
enzyme (data not shown). However, expression in E. coli
allowed the production of sufficient recombinant MAR1 protein to
generate anti-MAR1 polyclonal antibodies in rabbits.
To circumvent the low specific activity of the bacterial expressed
MAR1, an epitope-tagged (FLAG tag) version of the MAR1 protein was
expressed in L. tarentolae cells (see "Experimental Procedures"). The recombinant protein was purified using an
antibody-affinity column (Kodak). This expression approach yielded
apparently homogeneous MAR1 protein as determined by SDS-polyacrylamide
gel electrophoresis (Fig. 6A), as well as by Western
analysis using either anti-MAR1 antiserum or anti-FLAG antibody (Kodak)
(Fig. 6B). The anti-MAR1 antiserum recognized in a total
cell lysate of L. tarentolae a single band of the identical
gel mobility as the biochemically purified MAR1 and the recombinant
MAR1 proteins (Fig. 6B, lane 3).
The recombinant epitope-tagged protein expressed in L. tarentolae had a specific activity (160 ± 1.7 nmol/min/mg)
and a cleavage specificity that was indistinguishable from the native
enzyme (Fig. 3C), confirming that the MAR1 gene encodes the
MAR1 nuclease.
MAR1 Is a Single Copy Gene That Maps to an 820-kb Chromosome and
Has a Transcript of 1.3 kb--
The MAR1 gene was analyzed
by Southern blot hybridization of digested genomic DNA. L. tarentolae DNA was digested with a variety of restriction enzymes
and the blots hybridized with the full-length MAR1 gene
(data not shown). The enzymes, EcoRI, BamHI, and
HindIII, which do not cleave within MAR1,
generate a single DNA fragment that hybridizes with the MAR1
probe. The enzymes, BglII, HinfI, and
NcoI, which cleave once within the MAR1 open
reading frame, generated two DNA bands with the MAR1 probe.
BglII also cleaves at the 5' end of the gene leaving a
short fragment that was not detected. Double digestions also generated
patterns consistent with MAR1 being a single copy gene. The
restriction map of MAR1 is shown in Fig.
7A.
The MAR1 gene was localized to a 820-kb chromosome of
L. tarentolae by CHEF gel electrophoresis and Southern
analysis (Fig. 7C). A transcript of 1.3 kb is shown by the
Northern analysis in Fig. 7C.
MAR1 Does Not Contain a Cleavable Mitochondrial Targeting
Signal--
Analysis of the first 18 amino acids at the N-terminal of
MAR1 revealed characteristics compatible with other signal sequences reported in the literature. The presence of Arg, Leu, Ser, and Ala, the
presence of at least one Thr and one Lys, and the absence of Glu and
Asp residues are all consistent with this serving as a mitochondrial
targeting sequence (Fig. 5). Moreover, the N-terminal 18 amino acids of
MAR1 show the characteristic amphipathic pattern of known importation
signal sequences (data not shown).
Three genes were identified as possible homologues of MAR1: C. elegans (GenBankTM accession number Z69637), B. subtilis (GenBankTM accession number P37532), and
Archaeoglobus fulgidus (GenBankTM accession
number AE000943). These sequences are unidentified open reading frames.
The alignments to the MAR1 sequence are shown in Fig. 8, A
and B. The amino acid
alignments showed a high degree of similarity of the MAR1 sequence with
the C. elegans and A. fulgidus sequences (Fig.
8A). Moreover, the presence of an amphipathic helix at the N
terminus of C. elegans and its absence in the A. fulgidus is consistent with this N terminus sequence representing a mitochondrial importation signal in C. elegans as well.
The statistical significance of the alignments are shown in Fig.
8C; these values suggest that the alignments of the MAR1
sequence with the C. elegans and the A. fulgidus
are highly significant. The alignment of the MAR1 sequence with the
B. subtilis sequence is limited to the short region shown
boxed in Fig. 8B, but the statistical
significance of this alignment is high. The B. subtilis sequence is a homologue of the isochorismatase gene family, but a
comparison of the MAR1, C. elegans, and A. fulgidus polypeptide sequences with several members of the
isochorismatase gene family did not provide evidence that these genes
belong to that family (data not shown).
MAR1 is the first mitochondrial endoribonuclease purified from a
kinetoplastid protozoan. Isolation of this enzyme was facilitated by
its high relative thermal stability, which allowed the use of an
initial heating step to remove a large amount of protein prior to
chromatography on three columns. The purified MAR1 has an absolute
requirement for divalent cations and migrated in an SDS acrylamide gel
as a 22-kDa band. This enzyme may correspond to previously described
mitochondrial endoribonucleases from L. tarentolae and
T. brucei (17, 18). The L. tarentolae nuclease activity had an estimated molecular mass between 10 and 30 kDa, had an
absolute divalent cation requirement, and showed a major cleavage site
of pre-edited Cyb mRNA 2 nucleotides upstream of editing site 1 in
addition to several other cleavages throughout the PER (18). However,
the L. tarentolae activity also showed a stimulation by
heparin or by digestion of the crude lysate with proteinase K and an
inhibition by adenylate nucleotides or GTP. The T. brucei
nuclease activity showed a relatively high thermal stability, had a
specificity for sites within the PER's of Cyb, cytochrome oxidase II,
and cytochrome oxidase III mRNA substrates, and did not cleave
within the fully edited regions of the same RNAs (17). Both activities
were only characterized from crude mitochondrial lysates.
Purified MAR1 cleaved a synthetic pre-edited Cyb substrate at
approximately the same location as the previously described activities,
but showed additional cleavages 5' and 3' of the PER. The MAR1 enzyme
also did not distinguish between pre-edited and fully edited Cyb
mRNA substrates and was unaffected by the addition of heparin and
was sensitive to proteinase K (data not shown). Although these
differences could mean that MAR1 and the previously described nucleases
are different enzymes, they could also reflect structural differences
between the Cyb RNA substrates used in the cleavage reactions and could
be due to the fact that the previous nuclease activities were detected
and characterized in crude mitochondrial lysates, whereas this study
was performed both with a highly purified and a recombinant enzyme.
The demonstration that the affinity-purified recombinant FLAG-tagged
MAR1 protein expressed in L. tarentolae exhibited nearly identical specific activity and cleavage specificity to that of the
native enzyme provides definitive evidence that the MAR1 gene encodes
the MAR1 enzyme.
The cleavage of the Cyb and ND7 pre-edited mRNA substrates by MAR1
occurred in a gRNA-independent fashion. Interestingly, when increasing
concentrations of a cognate gRNA were annealed to the ND7 substrate
mRNA prior to the cleavage reaction, an inhibition of the cleavage
adjacent to the first editing site was observed. The presence of
several gRNA-independent endoribonucleases in a mitochondrial extract
from T. brucei has been described previously (14, 15, 31).
One of these activities co-sedimented with other editing activities in
glycerol gradients, but was separable from the
gRNA-dependent nuclease believed to be the editing-specific nuclease (14). This activity required DTT to function in
vitro and cleaved pre-edited mRNAs in the PER. MAR1 shows a
similar cleavage and fractionation behavior as the DTT-requiring
enzyme, but has no requirement for DTT (data not shown).
The lack of specificity for pre-edited mRNAs and the lack of a
gRNA-dependent cleavage at ES1 both suggest that MAR1 may
not be involved in RNA editing. However, it should be pointed out that
additional, yet undetermined, specificity factors could confer gRNA-dependence to MAR1 and this must remain an open question. Also, an
in vitro RNA editing-like activity independent of gRNA but
dependent on the secondary structure of the mRNA substrate has been
described in mitochondrial extracts from L. tarentolae (32,
33), and an involvement of MAR1 in this process still remains a possibility.
MAR1 is a mitochondrial protein that lacks a cleavable N-terminal
mitochondrial targeting sequence. We presented evidence that the 18 N-terminal amino acids represent a noncleaved signal sequence, but this
must be confirmed by direct experimentation. Cytochrome
c1 is the only other known example of a
trypanosome protein targeted to the mitochondrion without a cleavable
presequence (34). The MAR1 protein sequence is fairly conserved in
evolution, as homologues were found in a eukaryote and an
archaebacterium, and a conserved motif was found in a eubacterial
protein. The homologies, however, could not be used to determine a
function, as the sequences are unidentified reading frames.
As stated above, the possibility of MAR1 being involved in RNA editing
remains open, but it is equally likely that MAR1 could be involved in
mitochondrial RNA turnover. The expression of an epitope-tagged MAR1 in
E. coli and in L. tarentolae has permitted the
generation of an anti-MAR1 antiserum. Use of this immune reagent together with a detailed kinetic characterization of the cleavage reaction should help answer further questions about the specificity, substrate recognition, and mechanism of action of this nuclease.
We thank members of the Simpson laboratory
for helpful discussions and advice. We also thank Dr. J. Lake for
helpful discussions on the sequence analysis.
*
This work was supported in part by Grant AI09102 from the
National Institutes of Health (to L. S.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF083881.
The abbreviations used are:
Cyb, cytochrome
b; gRNA, guide RNA; MSB, mitochondrial storage-breakage
buffer; CHAPS, 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; DTT, dithiothreitol; RT-PCR, reverse transcription-polymerase chain
reaction; slRNA, spliced leader RNA; bp, base pair(s); kb, kilobase(s); CHEF, contour-clamped homogeneous field electrophoresis; PER, pre-edited region.
Purification and Characterization of MAR1
A MITOCHONDRIAL ASSOCIATED
RIBONUCLEASE FROM LEISHMANIA
TARENTOLAE*
,§¶
Howard Hughes Medical Institute, the
§ Department of Molecular,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
70 °C in mitochondrial storage-breakage buffer (MSB)
containing 50 mM HEPES (pH 7.5), 50 mM KCl, and
10% glycerol. Protein assays were performed using the BCA assay (Pierce).
70 °C or immediately used for MAR1
purification and analysis. Storage at 70 °C for long periods of
time (1-3 months) produced no loss of nuclease activity.
70 °C. Peak fractions from the
different chromatographic steps were electrophoretically separated in
10% Tricine/SDS gels and silver-stained (Bio-Rad).
-32P]pCp using T4 RNA ligase (Life Technologies,
Inc.). Alternatively, the mRNAs were dephosphorylated with calf
intestinal alkaline phosphatase in calf intestinal alkaline phosphatase
buffer as described by the manufacturer (Life Technologies, Inc.) and
5' end-labeled by incubation with [
-32P]ATP and T4
polynucleotide kinase for 1 h at 37 °C, in T4 polynucleotide kinase buffer (Life Technologies, Inc.). Once labeled, the RNAs were
gel-purified by electrophoresis in 7 M urea/8% acrylamide, and the bands were visualized by exposure of the gel to x-ray film. The
RNA was recovered by elution for 10 h at 4 °C in 400 µl of
300 mM sodium acetate, 1 mM EDTA buffer. The
labeled RNA was precipitated with 1.2 ml of ethanol at 20 °C for
15 min. The pellets were washed twice with 70% ethanol, dried under
vacuum, and suspended in TE buffer (10 mM Tris, 1 mM EDTA (pH 8.0)).
competent cells.
Insert-containing plasmids from several E. coli clones were sequenced.
cells.
-D-thiogalactopyranoside-responsive
promoter. E. coli BL21 cells were grown in 2 × YT
medium, and recombinant protein expression was induced by the addition
of isopropyl--D-thiogalactopyranoside (2 mM
final concentration) as described in the Qiagen manual. The His-tagged
recombinant MAR1 protein was purified by metal-chelate chromatography (Qiagen).
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Effect of preincubation at different
temperatures on the MAR1 reaction and divalent cation
requirements. A, constant amounts of unclarified
mitochondrial detergent lysates were heated in buffer for 20 min at
different temperatures. Samples were quenched in ice, radioactively
labeled substrate was added, and the samples were incubated at 27 °C
for 1 h. The activity at 27 °C was taken as the 100% value.
B, divalent cation requirement for the MAR1 reaction.
Reactions were performed with constant concentrations of MAR1 (50 ng),
saturating concentrations of substrate (500 nM), and
increasing concentrations of the chloride salts of Mg2+
( 
), Mn2+ (
), Zn2+ (
), and
Ca2+ (
). Relative activity, expressed as the percentage
of the substrate cleaved in 1 h at 27 °C, was plotted
versus the different cation concentrations.
Purification of MAR1
20 min
clarified supernatant of the CHAPS lysate (as described under
"Experimental Procedures"). S-100 is the supernatant after
centrifugation of the crude fraction at 100,000 × g
for 1 h. Mono Q, Superose 12, and Mono P represent the pooled
peaks of nuclease activity from each chromatography step. A unit is
defined as 1 nmol of substrate RNA cleaved per min. N/A, not available.
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Fig. 2.
Chromatography of MAR1. A,
Mono Q anion exchange chromatography of the heated S-100 supernatant.
The column was equilibrated with QA buffer and developed with a linear
gradient of QB buffer. The shaded area represents the
elution of the peak of cleavage activity. B, chromatogram of
the Superose 12 fraction on a Mono P chromatofocusing column. The
shaded area represents the peak of cleavage activity. The
A280 of each fraction is plotted. The peak of
activity corresponds to pH 6.5-6.8. C, silver-stained 10%
SDS-acrylamide gel of various fractions in the purification of MAR1.
S-100 refers to the heat-treated supernatant. Mono Q, Superose 12, and
Mono P refer to the peak fractions at each chromatographic step. The
arrow ( 
) denotes the position of the 22-kDa MAR1
band.
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Fig. 3.
Mapping of the MAR1 cleavage sites on
pre-edited ND7 and pre-edited and fully edited Cyb mRNA
substrates. A, synthetic ND7 pre-edited mRNA was 5'
end-labeled with [ -32P]ATP. The labeled RNA was used
as a substrate for MAR1 cleavage in the presence (lanes
9-11) or absence of gRNA (lanes 6-8), and in the
presence (lanes 6-11) or absence of MAR1 (lane
1). Lanes 2-5 show the product of a sequencing ladder
generated with the sequence-specific nucleases T1RNase (G), U2 RNase (A), Phy M RNase (A + U), and B. cereus RNase (U + C). "ES1" and
"ES2" refer to the relative migration of fragments
cleaved at editing sites 1 and 2. B, 3' end-labeled
synthetic pre-edited Cyb (PE) mRNA (lanes 1 and 2) and fully edited Cyb (FE) mRNA
(lanes 3 and 4) were incubated in the presence
(lanes 2 and 4) and absence (lanes 1 and
3) of MAR1. (Similar results were obtained using 5'
end-labeled substrates.) Arrows indicate major cleavage
sites. The length in nucleotides (nt) of various markers is
shown on the left. An asterisk denotes the
position of the radioactive label on the various substrates. The
locations of the PER and the cleavages within the 3'-unedited region
are indicated by brackets. The reactions were separated on a
7 M urea/10% acrylamide gel. C, FLAG-tagged
recombinant MAR1 enzyme, affinity-purified from transfected L. tarentolae cells, was used with the identical PE and FE RNA
substrates as in B.
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Fig. 4.
Kinetic analysis of the MAR1 cleavage
reaction. Increasing concentrations (6-80 nM) of
5'-labeled pre-edited ND7 mRNA (A), pre-edited Cyb
(B), and fully edited Cyb mRNA (C) were
incubated with 10 ng (9.2 µM) MAR enzyme at 27 °C for
30 min. The fraction of the various concentrations of input RNA cleaved
was determined by PhosphorImager analysis of the dried gels. Fraction
cleaved (cleaved product/cleaved product + uncleaved × 100/reaction time) was used to calculate the amounts of cleaved product
made per min. Double-reciprocal plots of velocity versus
substrate concentration were used to calculate the
Km and Vmax for the MAR1
enzyme under initial velocity conditions.
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Fig. 5.
Nucleotide sequence and predicted amino acid
sequence of the MAR1 gene from L. tarentolae. The 3-kb EcoRI-BamHI
cloned genomic fragment was sequenced. A segment of 1298 bp of this
fragment is shown. Nucleotides within the protein coding region of
MAR1 are numbered positively from the Met start
codon (boxed ATG), and the predicted amino acid residues of
MAR1 are numbered positively starting from the first Met. A
termination codon (TAA) is indicated in bold at position
+579. The predicted mitochondria importation signal sequence is
underlined. Hydrophobic amino acids are indicated by an ,
and positively charged amino acids are indicated by a +. Nucleotides
within the 5'-untranslated region are numbered negatively
starting from the initiation ATG codon from MAR1. The
nucleotides that correspond to the splice acceptor sites of
MAR1 transcript are indicated (SAS-1 and SAS-2).
Polypyrimidine tracks 5' to each splice acceptor site are
boxed. The two polyadenylation sites identified in the
MAR1 transcript are indicated at positions +1003 and
+1143.
33 from the adenylate residue of the predicted methionine translation
initiation codon (boxed in Fig. 6) and is preceded by a 10 nucleotide
polypyrimidine track at positions 57 to 48. The second splice
acceptor site (SAS-2) is located at position 123, with an upstream
polypyrimidine track at positions 157 to 149 (Fig. 5). Primer
extension assays of total RNA using the 5' end-labeled S-2474 primer
revealed that SAS-1 is used for splicing of the slRNA in 60% of the
transcripts, and SAS-2 is used in the remaining 40% (data not shown).
RT-PCR analysis of the 3' end of MAR1 revealed two polyadenylation
sites, one at position +1003 and another at position +1143, 424 and 564 nucleotides, respectively, from the second adenylate residue of the
predicted TAA termination codon. These results would predict a
transcript ranging from a maximum of 1305 nucleotides to a minimum of
1075 nucleotides (not including the length of the poly(A) tail).
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Fig. 6.
Expression of the FLAG-tagged MAR1
in L. tarentolae cells. A, electrophoretic
separation, on a 10% acrylamide/Tricine-SDS gel, of various fractions
from an anti-FLAG affinity column. The gel was stained with Coomassie
Brilliant Blue. Lane 1, cell-free extract prior to loading
on the affinity column. Lane 2, the eluant fraction from the
column loading. Lane 3, the column was washed with "low
salt" buffer 50 mM Tris (pH 8.0), 100 mM
NaCl. Lane 4, "high salt" wash with 50 mM
Tris (pH 8.0), 400 mM NaCl. Lane 5, 0.1 M glycine (pH 4.0) elution. M denotes the size
markers used during electrophoresis. B, Western blot of the
recombinant MAR1 expressed in L. tarentolae cells and the
native MAR1. In lanes 1 and 2, recombinant MAR1
was electrophoretically separated on 10% tricine-SDS acrylamide gels,
and the gels were electroblotted onto nitrocellulose membranes and
probed with polyclonal antiserum raised against the E. coli-expressed protein (lane 1) or with monoclonal
antibody against the FLAG epitope (Kodak) (lane 2). An
identical Western analysis of total cell extract from L. tarentolae using the polyclonal MAR1 antiserum is shown in
lane 3. The position of the MAR1 protein is indicated by a
double arrow.
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Fig. 7.
Molecular characterization of the MAR1
gene from L. tarentolae. A, schematic
representation of the MAR1 locus restriction fragment map.
The probe used in the Southern and Northern blots, as well as in the
CHEF gel hybridization experiments, is indicated. B,
Southern blot of a CHEF gel electrophoresis of L. tarentolae
chromosomal DNA. A band of approximately 820 kb hybridized with the
MAR1 probe. The size markers are S. cerevisiae
chromosomes (Promega). C, Northern blot hybridization of
L. tarentolae total RNA with the probe indicated in
A. A single transcript of approximately 1.3 kb is detected.
RNA size standards are indicated on the left.
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Fig. 8.
Alignment of MAR1 amino acid sequence with
data base sequences. A, the predicted polypeptide
sequence of MAR1 is aligned with the C. elegans
(GenBankTM accession number Z69637) and A. fulgidus (GenBankTM accession number AE000943)
sequences. The predicted N terminus signal sequences of MAR1 and the
C. elegans sequence are underlined. The consensus
of the alignment is indicated. "*" indicates amino acid identities;
":" and "." indicate conserved substitutions. The predicted
secondary structure fold (above 90% certainty) utilized as a guide to
refine the sequence alignment is shown: H, -helices; L, loops, and
B, -sheets. B, domain of the multiple sequence alignment,
including the isochorismatase sequence from B. subtilis
(GenBankTM accession number P37532) (boxed
region in A). C, table of S.D. values
resulting from a Monte Carlo statistical analysis of the different
sequences (>5 S.D. units is considered a significant match).
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence and reprint requests should be
addressed. Tel.: 310-825-4215; Fax: 310-206-8967; E-mail:
simpson{at}hhmi.ucla.edu.
REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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