From the Department of Plant Pathology, University of Kentucky, Lexington, Kentucky 40546-0091
Received for publication, August 23, 2000, and in revised form, October 18, 2000
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ABSTRACT |
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We have cloned and sequenced a novel alcohol
oxidase (Hv-p68) from the filamentous fungus
Helminthosporium (Cochliobolus) victoriae that copurifies with mycoviral double-stranded
RNAs. Sequence analysis revealed that Hv-p68 belongs to the large
family of FAD-dependent glucose methanol choline
oxidoreductases and that it shares significant sequence identity
(>67%) with the alcohol oxidases of the methylotrophic yeasts.
Unlike the intronless alcohol oxidases from methylotrophic yeasts, a
genomic fragment of the Hv-p68 gene was found to contain
four introns. Hv-p68, purified from fungal extracts, showed only
limited methanol oxidizing activity, and its expression was not induced
in cultures supplemented with methanol as the sole carbon source.
Northern hybridization analysis indicated that overexpression of Hv-p68
is associated with virus infection, because significantly higher Hv-p68
mRNA levels (10- to 20-fold) were detected in virus-infected
isolates compared with virus-free ones. We confirmed by Northwestern
blot analysis that Hv-p68 exhibits RNA binding activity and
demonstrated that the RNA-binding domain is localized within the
N-terminal region that contains a typical ADP-binding
It has recently become increasingly clear that cellular factors
play important roles in the transcription and replication of RNA
viruses (1). The double-stranded RNA
(dsRNA)1 genomes of the two
mycoviruses (2, 3) known to infect the plant pathogenic fungus
Helminthosporium victoriae (teleomorph: Cochliobolus
victoriae; synonym: Bipolaris victoriae) consists of
two genes that encode a capsid protein (CP) and an
RNA-dependent RNA polymerase (RDRP). The CP and RDRP genes
are either present on the same dsRNA segment, as in the case of the
totivirus H. victoriae 190S virus, or on two separate dsRNA
segments, in the case of the putative chrysovirus H. victoriae
145S virus (Hv145SV). It is thus not surprising that these viruses
subvert host proteins for their own use. We have previously reported
that host enzymes (a protein kinase and a protease) are involved in
post-translational modification of the Hv190SV CP (4-6).
H. victoriae 190S virus (Hv190SV) has been extensively
studied (2-9), and is classified as a definitive member of the genus Totivirus in the family Totiviridae (10,
11). The Hv145SV, on the other hand, has only been subjected to limited
biochemical characterization (2). Because of similarity in size and
number of dsRNA segments between the Hv145SV and viruses in the genus Chrysovirus in the family Partitiviridae, the
Hv145SV was tentatively classified as a member of the genus
Chrysovirus (12). We have recently (13) isolated a cellular
protein, Hv-p68, that copurifies with viral dsRNA from H. victoriae isolates infected with both Hv190SV and Hv145SV.
Additionally, Hv-p68 was demonstrated to be present as a minor
component in the viral capsids (13). Our initial biochemical
characterization studies of Hv-p68 indicated that it occurs in
vivo as an octamer and that it is consistently present in higher
levels in virus-infected H. victoriae isolates than in
virus-free ones. We have also demonstrated by a gel retardation assay
that Hv-p68 has RNA binding activity (13).
In the present study, we report the isolation and complete nucleotide
sequence of a cDNA clone of Hv-p68. We present evidence that Hv-p68
belongs to the FAD-dependent GMC family of oxidoreductases and that it has high sequence similarity to the alcohol oxidases of
methylotrophic yeasts. Furthermore, we show that overexpression of
Hv-p68 is associated with virus infection and that the RNA-binding domain of Hv-p68 is localized in the N-terminal region that contains a
canonical ADP-binding Fungal Isolates--
Three H. (Cochliobolus) victoriae isolates that differ in
their virus content were used. Isolate A-9 (ATTC 42018), a diseased isolate known to contain both Hv190SV and Hv145SV (1), was routinely
used as a source for virions and Hv-p68. A single conidial isolate of
H. victoriae isolate B-2 (ATCC 42020), designated B-2ss, has
recently been demonstrated to be devoid of virus and was used as a
representative of a cured fungal isolate (13). The virus-free H. victoriae isolate 408, used in previous virus transmission studies
(14), was used as an example of a naturally occurring virus-free
isolate. Isolates of C. heterostrophus, C. zeicola, and C. sativum, provided by M. Carson (North
Carolina State University), were used in Southern screening for the
Hv-p68 gene. DNA from the filamentous fungus P. chrysogenum (ATCC 9480), Saccharomyces cerevisiae
(strain 717; provided by Reed Wickner) and Schizosaccharomyces pombe (strain SP-Q01; Stratagene) were also included in the
Southern analysis.
Isolation of mRNA and cDNA Synthesis--
Total RNA was
isolated from 4-day-old stationary cultures of H. victoriae
strain A-9 by the guanidinium isothiocyanate/phenol method (15).
Polyadenylated RNA was purified from total RNA by oligo(dT)-Sepharose
affinity chromatography (Amersham Pharmacia Biotech). First and second
strand cDNA were synthesized from poly(A) RNA using the SuperScript
cDNA synthesis kit (Life Technologies, Inc.).
PCR Amplification of Hv-p68 cDNA Sequences--
Forward and
reverse degenerate primers corresponding to the N-terminal sequence of
the native Hv-p68 protein (primer P1; see Fig. 1) and to
amino acid sequencing data of an internal tryptic peptide (primer
P2; see Fig. 1) were used along with the double-stranded cDNA
synthesized to fungal mRNA (Hv-cDNA) to prime the amplification of an Hv-p68 cDNA fragment. Amplification reactions were carried out using Platinum High Fidelity Taq DNA polymerase (Life
Technologies, Inc.) and cycling parameters specified for TD-PCR
(94 °C for 4 min, 94 °C for 1 min, 60 °C ( Construction of a cDNA Library--
Poly(A)+ RNA
isolated from a 3-day-old stationary culture of isolate A-9 of H. victoriae was used to generate a cDNA library in the lambda
ZipLox vector following the procedures for the SuperScript Lambda
cloning system (Life Technologies, Inc.). The library was packaged
using Gigapack II Gold (Stratagene) and amplified once to a high titer
(8 × 1010 plaque-forming units/ml) in
Escherichia coli cells. Hv-p68 lambda clones were isolated
by screening ~800,000 recombinant phages with a
32P-labeled Hv-p68 probe generated by nick-translation
(Life Technologies, Inc.) of the 1.5-kbp DOP-PCR product. Replica lifts
(Hybond N, Amersham Pharmacia Biotech) from plated phage were
hybridized for 14-16 h at 42 °C in 50% formamide/hybridization
solution (50% formamide/1× Denhardt's solution/5× SSC/0.5% SDS/20
mM sodium phosphate, pH 7.0) and washed under conditions of
high stringency. Pure lambda clones were obtained by two successive
rounds of screening with the 32P-labeled 1.5-kbp cDNA
probe. Four independent lambda clones were selected for in
vivo excision into the cloning vector pZL1 as described by the
manufacturer (Life Technologies, Inc.), and subjected to automated sequencing.
Nucleotide Sequencing and Analysis--
All sequencing was
performed by dideoxy-termination sequencing using the
Rhodamine-Terminator sequencing kit (ABI) and an ABI600 automated
sequencer. Automated sequencing was performed on both strands with
universal primers and gene-specific "walking" primers. Sequence
homology searches of GenBankTM, Swissprot, and EMBL data bases were
conducted using the BLAST and FASTA programs (16, 17). Paired and
multiple sequence alignments were carried out with the programs
Best-Fit, GAP, PILEUP, and PRETTY (Wisconsin GCG software package
(18)). Motif and signature pattern searches were conducted using the
program Motif (19). Sequence alignments and phylogenetic analysis were
performed using the program ClustalW (20). Trees generated by ClustalW
were displayed using TreeView (21). Predictions for protein sorting
signals and subcellular localization sites on the deduced amino acid
sequence were performed using PSPRT II (22).
Expression of the Hv-p68 ORF in Bacteria and Affinity
Purification of the C-terminal His-tagged Protein--
A cDNA
corresponding to the coding region of Hv-p68 was obtained by PCR
amplification using gene-specific primers and one of the isolated
lambda Hv-p68 cDNA clones as a template. Primer sequences
corresponding to the 5'- and 3'-ends of the Hv-p68 ORF and containing
restriction-enzyme site sequences for cloning were: pET-p68-EcoRI FOR,
5'-CCGGAATTCGACGATCCCGGACGACGAGGTTGATATTATCG-3' and
pET-p68-NotI REV,
5'-AATATTCTTAGCGGCCGCCAAGCGCGATAATCCAGCAATC-3'. Amplification reactions were carried out using standard PCR conditions and Platinum High Fidelity Taq DNA polymerase (Life
Technologies, Inc.). The PCR-amplified product (~2 kbp) was
gel-purified by agarose gel electrophoresis followed by extraction with
GeneClean (Bio101), digested with EcoRI-NotI, and
ligated into an EcoRI-NotI-digested pET22(b)+
vector (Novagen). The construct, pETp68, was used to transform E. coli strain BL21(DE3) cells according to the manufacturer's instructions (Novagen). For purification of the bacterially expressed Hv-p68, 100-ml cultures of transformed BL21(DE3) cells were grown in LB
media at 37 °C to a density of 1.0 A600 nm.
Protein was expressed by induction with 0.5 mM
isopropyl-1-thio- Bacterial Constructs for Expression of N-terminal and C-terminal
Deletion Mutants of Hv-p68--
Constructs were generated in the
bacterial expression vector pET22(b)+ containing large deletions
at either the N- or C-terminal sequence of the Hv-p68 coding region. A
construct for expression of a 271-amino acid N-terminal deletion mutant
was generated by subcloning an SstI-NotI
restriction fragment from construct pETp68 (corresponding to nt
positions 813-2111 in the Hv-p68 cDNA; see Fig. 1) into a
similarly digested pET22(b)+ vector. A 160-amino acid C-terminal
deletion construct was generated by subcloning the 1.5-kbp
EcoRI-NotI fragment from construct pZp68-1.5kbp
(corresponding to nt positions 4-1512; see Fig. 1) into the
EcoRI-NotI-digested sites of pET22(b)+. 50-ml
cultures of BL21(DE3) cells transformed with the full-length Hv-p68
(pETp68) and the N- and C-terminal truncated constructs were grown in
LB media at 37 °C to a density of 1 A600 nm.
Protein was expressed by induction with 1 mM
isopropyl-1-thio- Nucleic Acid Isolation and Hybridization--
Total RNA was
isolated by a modification of the procedure of Chomczynski and Sacchi
(15). H. victoriae isolates A-9, B-2ss, and 408 were grown
in stationary cultures in Fernbach flasks containing 200 ml of potato
dextrose broth medium supplemented with 0.5% yeast extract (PDBY) for
8 days at room temperature. For the induction studies, mycelium from
H. victoriae isolate B-2ss was collected by centrifugation
(3000 × g, 10 min) and rinsed with minimal medium (MM)
supplemented with 1% glucose (23). The mycelium was homogenized in a
blender, and the homogenate was transferred to Fernbach flasks containing MM supplemented with 1% dextrose and grown for 3 days on a
shaker incubator at 22 °C. The mycelium was harvested and rinsed
twice with MM, homogenized, and transferred to flasks containing fresh
MM supplemented with either 1% glucose or 1% methanol and grown on a
shaker incubator at 22 °C for 3 days. Mycelia from PDBY and MM
cultures were collected by straining through two layers of Miracloth
(Calbiochem) and pulverized in liquid nitrogen and 100 ml of a
guanidinium-denaturing solution/water-saturated phenol/2 M
sodium acetate buffer, pH 4.0 (48:47:5), added (per 10 g of wet
tissue). The suspension was incubated at room temperature for 15-20
min and centrifuged at 10,000 × g for 15 min, and the supernatant was extracted by addition of 0.2 volume of chloroform (20 ml). The aqueous phase was re-extracted with chloroform/isoamyl alcohol
(24:1), and the RNA was precipitated by addition of 1 volume of
isopropanol. The RNA pellets were resuspended in diethyl pyrocarbonate
treated water and subjected to two rounds of high salt-isopropanol
precipitation at room temperature (0.5 volume of 1.2 M
NaCl/0.8 M sodium citrate and 0.5 volume of isopropanol), followed by precipitation in 2 M LiCl (4 h at 4 °C). For
Northern hybridization, 25 µg of the total RNA was electrophoresed on
1% agarose-formaldehyde gels and transferred to a nylon membrane (Hybond N, Amersham Pharmacia Biotech) by alkaline downward transfer (TurboBlotter, Schleicher & Schuell), and the blot was subjected to UV
cross-linking. Blots were hybridized with
[ Southern Analysis--
Fifteen micrograms of the restriction
enzyme-digested genomic DNA was electrophoresed on a 0.8%
agarose gel (at 80 V for 9-10 h), and the DNA was transferred
to a nylon membrane (Hybond N, Amersham Pharmacia Biotech) by neutral
downward transfer (20× SSC; TurboBlotter, Schleicher & Schuell), and
the blot was subjected to UV cross-linking. Blots were hybridized with
an [ Alcohol Oxidase Assay--
Alcohol oxidase activity in Hv-p68
preparations from H. victoriae and the bacterially expressed
Hv-p68 was determined by a spectrophotometric assay, which measures the
rate of conversion of methanol to hydrogen peroxide (24). Production of
hydrogen peroxide after addition of methanol to the reaction was
determined by monitoring the change in absorbance at 405 nm (at
25 °C) over a 5-min period using
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (A-1888, Sigma) as
substrate and horseradish peroxidase (P-8250, Sigma). Commercially
purified Pichia pastoris alcohol oxidase (E.C. 1.1.3.13;
A2404, Sigma) was used in parallel enzymatic assays for comparison.
Protein content was measured using a Bio-Rad protein determination
reagent. Activity was given as Radiolabeled in Vitro Transcripts--
Radiolabeled transcripts
(riboprobes) were synthesized by in vitro transcription from
a pUC-construct pT7Hv190S, containing a full-length cDNA of the
Hv190S dsRNA (3), and pZ-2-28 containing a near-full length
cDNA of the Hv145S dsRNA-4 in the pZErO vector. The
positive-strand transcripts were generated by incubation of pT7Hv190S
and pZ-2-2, previously linearized with BamHI and
NotI, respectively, with T7 RNA polymerase (Stratagene) at
37 °C for 60 min in the presence of [ Northwestern Blotting Analysis--
Proteins were separated on
10% SDS-polyacrylamide gels and transferred to a nitrocellulose
membrane (BA85, 0.45 µm, Schleicher & Schuell) using a wet transfer
apparatus (Bio-Rad). The membranes were then washed three times at room
temperature, 20 min each time, in buffer A (10 mM Tris-HCl,
pH 7.5, 1 mM EDTA, 50 mM NaCl, 0.1% Triton
X-100, 1× Denhardt's reagent) to renature the proteins. Blotted
proteins were probed by incubating with the 32P-labeled
transcripts (105 cpm) in the same buffer for 1 h at
room temperature. The blots were then washed three times for 2 min each
using the same buffer, air-dried, and autoradiographed. After
autoradiography, blots were rinsed briefly in Tris-buffered
saline-Tween 20 (TBS-T) and incubated for 1 h in
blocking buffer (TBS-T, 5% nonfat dry milk). Blots were processed for
immunoblotting using the polyclonal antibodies against Hv-p68 (13) and
goat anti-rabbit IgG-alkaline phosphatase conjugate as secondary
antibodies. Nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl
phosphate (Promega) were used as substrates for colorimetric detection.
Isolation and Characterization of Hv-p68 cDNA
Clones--
cDNA clones for the Hv-p68 gene were
isolated by a combination of DOP-PCR and conventional library
screening. The amino acid sequencing data previously obtained by Edman
degradation analysis (13) were used to design two sets of degenerate
primers for DOP-PCR. Mixed pools of oligonucleotides corresponding to
the N-terminal sequence of the native protein (primer P1;
Fig. 1) and to an internal tryptic
peptide (primer P2; Fig. 1) were used to direct the
amplification of an Hv-p68 PCR product with Hv-cDNA as a template.
Amplifications using conditions for TD-PCR yielded a single major PCR
product ~1.5 kbp in size. This product was blunt-end-cloned into a
pZErO vector (Invitrogen) and subjected to automated sequencing.
The sequencing data for the 3'-end region of the 1.5-kbp fragment was
used to design a sequence-specific primer for amplification of the
3'-end region of the Hv-p68 cDNA. A 750-bp PCR product
corresponding to the 3'-end region of Hv-p68 was amplified using
Hv-cDNA as template and the sequence-specific primer, P3 (Fig. 1),
and an oligo(dT) primer. The 750-bp PCR product hybridized strongly in
Southern blots probed with the 32P-labeled 1.5-kbp PCR
product (data not shown). The 750-bp product was cloned into the vector
pZErO and sequenced. The deduced amino acid sequence of the
PCR-amplified products matched perfectly the amino acid sequence data
derived from the N-terminal sequence of the gradient purified Hv-p68
protein as well as that from the internal peptide sequence (Fig. 1,
amino acid residues printed in boldface letters).
To validate the clones obtained by the DOP-PCR cloning procedure and to
isolate a full-length cDNA, we generated a cDNA library for
C. victoriae in lambda phage. A primary screening of
~800,000 recombinant phages from the amplified library with the
radiolabeled 1.5-kbp PCR product yielded four positive clones. These
clones were independently purified by two additional rounds of plating and screening with the radiolabeled probe, and the selected lambda clones were sequenced. The sequencing data generated from all four
lambda clones were consistent with that of the cDNA obtained through the DOP-PCR approach. Three of the four lambda clones had
cDNA inserts with 5'-end truncations of the coding region. The
fourth clone (2148 bp in length), however, was found to contain the
entire coding region (1998 nt, including the termination codon) and an
additional 37 nt derived from the 5'-untranslated region. The initiator
ATG (starting at nt position 1, Fig. 1) is present in a
favorable context according to Kozak (25)
(CAGAATGAC), and the region surrounding the ATG
agrees with the consensus sequence for filamentous fungi (26)
(5'-CAMMATGNC, where M = A or C; N = A, C, G, or T). That the
ATG at nt positions 1-3 corresponds to the translational start codon
is supported by the Edman degradation sequencing data of the N terminus
of Hv-p68 (Fig. 1). The complete nt sequence and deduced amino acid
sequence for the Hv-p68 cDNA is shown in Fig. 1. The coding region
of Hv-p68 codes for a 665-amino acid protein with a predicted molecular
mass of 74,251 Da. This value is in agreement with our estimate of 68 kDa, based on SDS-PAGE analysis (13).
Analysis of the deduced amino acid sequence of Hv-p68 revealed the
presence of three potential sites for N-linked glycosylation with consensus sequence NX(S/T) (Fig. 1,
underlined). A search for cellular sorting signals with the
program PSORT identified a PTS1 site at the C terminus of the deduced
amino acid sequence (the tripeptide SRL shown in
shaded box in Fig. 1) for translocation to the peroxisomal
compartment. The Hv-p68 sequence, however, lacks all the
well-established RNA-binding motifs (e.g. the RNP motif, RGG
box, etc. (27, 28)).
Although we have not isolated genomic clones that contain the entire
Hv-p68 gene, we obtained genomic sequence information from a
partial genomic clone generated by DOP-PCR amplification using the
degenerate primers P1 and P2 with C. victoriae genomic DNA
as a template. The single PCR product obtained (1.7 kbp) was blunt-end-cloned into the vector pZErO (Invitrogen) and
sequenced. Comparison of the sequence of the genomic fragment with the
corresponding sequence for the Hv-p68 cDNA indicated the coding
region is interrupted by four short introns ranging in size from 50 to
59 bp (Fig. 2). Interestingly, the first
intron interrupts the sequence of the coding region very close to the N
terminus of Hv-p68 between amino acid positions 6 and 7 (Fig. 2). All
four introns are in general agreement with the filamentous fungi
consensus sequence (29) for 5'-splice junctions (5'-GTDHSY; where
D = A, G, or T; H = A, C, or T; S = C or G; Y = C
or T) and 3'-splice sites (5'-YAG) as well as internal putative lariat
formation elements (5'-NNYTNAY; where N = any nt).
Sequence Comparisons--
Homology search of protein data bases
using the BLAST program revealed that the deduced amino acid sequence
of the Hv-p68 cDNA has significant similarity with members of the
large family of FAD-dependent GMC oxidoreductases (30). The
highest sequence identity scores were obtained with the alcohol
oxidases of the methylotrophic yeasts (with identities higher than
67%). A multiple alignment of the deduced amino acid sequences for
Hv-p68 and for those reported for methanol oxidases are given in Fig.
3. Hv-p68 shares the five signature
patterns (blocks A-E (19)) characteristic of the flavoproteins in the
GMC oxidoreductase superfamily (Fig. 3, shaded areas). Block
A comprises part of the FAD ADP-binding region with its typical
The FAD-dependent GMC oxidoreductases have been reported to
be evolutionarily related (30). The results of pairwise alignments, using the GAP program, of Hv-p68 and six members of the family of
FAD-dependent GMC oxidoreductases, including four alcohol
oxidases from methylotrophic yeasts are shown in Fig.
4A. Hv-p68 showed significantly high similarity and identity scores to the alcohol oxidases; the highest values were recorded for the AOX1 from P. pastoris with 75.5% similarity and 69.7% identity (Fig.
4A). Likewise, phylogenetic analysis showed that Hv-p68
forms a cluster with the alcohol oxidases from methylotrophic yeasts
that is strongly supported by bootstrap analysis (Fig.
4B).
Bacterial Expression and Immunological Verification of the Hv-p68
cDNA Clones--
The coding region of the Hv-p68 fused to a
C-terminal His-tag was expressed in the bacterial expression vector
pET22(b)+. The overexpressed protein was purified on a Ni-NTA column
and analyzed by SDS-PAGE and Western blotting. The bacterially
expressed protein reacted strongly with antibodies against Hv-p68 (Fig. 5A), thus providing
immunological verification for the Hv-p68 cDNA clone. The antiserum
to Hv-p68 also reacted with P. pastoris alcohol oxidase (a
commercial preparation from Sigma), but the intensity of the Western
blot band was clearly lower than that obtained with the homologous
antigen (Fig. 5A).
Localization of the RNA Binding Domain--
The RNA binding
activity of Hv-p68 has previously been demonstrated in gel-retardation
assays using Hv-p68 preparations purified from fungal extracts (13). To
localize the RNA-binding domain of Hv-p68, we compared the RNA binding
activity of bacterially expressed full-length and deletion mutants of
Hv-p68 ORF using Northwestern blotting analysis (this study). Hv-p68
purified from the fungus and the Ni-NTA-purified bacterially expressed
Hv-p68 were subjected to SDS-PAGE and blotted onto a nitrocellulose
membrane, and the blots were incubated with radiolabeled riboprobes
prepared from cloned cDNAs to Hv190SV or Hv145SV dsRNAs. As shown
in Fig. 5B, strong binding signals corresponding to binding
of the RNA probe to Hv-p68 were detected. Similar results were observed
using either the Hv190S or Hv145S riboprobe. The bands on the
autoradiograph corresponded to the prominent protein bands on Western
blots of the fungal- and the bacterially expressed Hv-p68, which
reacted with antibodies to Hv-p68 (Fig. 5A). The binding of
the P. pastoris AOX to the Hv190S and 145S riboprobes was
very weak (detectable only in an overexposed autoradiograph) compared
with that observed for either the fungal- or bacterially expressed
Hv-p68.
Data base searches with the deduced amino acid sequence of Hv-p68
indicated the absence of consensus sequences for the well-characterized RNA-binding motifs previously reported for RNA-binding proteins (e.g. RNP motif, RGG box, etc. (27, 28)). Earlier studies with several NAD+-dependent dehydrogenases and
oxidoreductases have revealed that these enzymes may also function as
RNA-binding proteins (36). The structural feature that these enzymes
have in common is a region containing a typical Functional Analysis--
The methanol oxidizing activity of Hv-p68
purified from fungal extracts and the Ni-NTA purified bacterially
expressed Hv-p68 was tested by spectrophotometric measurement of
hydrogen peroxide formation. A commercial preparation of P. pastoris alcohol oxidase was used as a control. The results of the
enzyme assay are shown in Table I.
Although Pp-AOX was highly active with calculated activity of 14 units/mg of protein, Hv-p68 exhibited relatively low activity. No
methanol-oxidizing activity was detected with the bacterially expressed
Hv-p68. We assayed several Hv-p68 preparations purified from fungal
extracts, including freshly purified preparations as well as
preparations that had been kept frozen for several months. Although the
enzymatic activity was generally low, preparations that have been
subjected to repeated freezing and thawing showed the weakest
activities (data not shown).
Hv-p68 Expression in Virus-infected and Virus-free H. victoriae
Isolates--
Northern hybridization analysis was performed with total
RNA isolated from 12-day cultures of the virus-infected isolate A-9 and
the virus-free isolates 408 and B-2ss (13). In Northern blots
hybridized with a radiolabeled Hv-p68-specific probe, an mRNA
~2.3 kb in size was detected in all three fungal isolates. The size
of mRNA (2.3 kb) is consistent with the estimated size for the
Hv-p68 cDNA (~2.1 kbp). Interestingly, the level of the Hv-p68
mRNA in the virus-infected isolate A-9 was 10- to 20-fold higher
than that in the virus-free isolates. This indicates that overexpression of Hv-p68 is associated with virus infection. The increased levels of Hv-p68 transcription are consistent with the higher
amounts of Hv-p68 that are normally associated with cultures of the
virus-infected isolate A-9 (13).
Expression of the alcohol oxidases of the methylotrophic yeasts is
tightly regulated at the transcriptional level by a dual mechanism of
repression by glucose and induction by methanol (33). We examined the
possibility that the Hv-p68 gene may be under similar
transcriptional control by growing the virus-free isolate B-2ss in a
shake culture for 72 h in a minimal medium containing either 1%
glucose or 1% methanol. The B-2ss cultures grown on a minimal medium
supplemented with 1% methanol as the sole carbon source showed little
or no apparent increase in mycelial mass compared with the cultures
supplemented with glucose, which showed significant growth during the
72-h period. Northern blot analysis with equivalent amounts of total
RNA (30 µg) isolated from these cultures revealed that the level of
Hv-p68 transcript was not significantly different between the glucose
and the methanol-supplemented cultures (Fig.
7B). Addition of methanol to
the culture medium, under our experimental conditions, did not appear
to influence the transcription rate of the Hv-p68 gene.
Distribution of the Hv-p68 Gene in Fungi--
We examined the
presence of the Hv-p68 gene or closely similar genes by
Southern hybridization analysis using genomic DNA isolated from various
fungal species and an Hv-p68 probe. The restriction profiles produced
with restriction enzymes known to cleave the DNA within the Hv-p68
coding region or elsewhere varied with different fungal species. All
Cochliobolus species tested contained the Hv-p68
gene or a closely similar gene (Fig. 8). The restriction profiles for C. victoriae isolate 408 were
different from those generated for C. victoriae isolates
B-2ss and A-9, suggesting that C. victoriae is probably not
monophylatic. No genes closely similar to Hv-p68 were
detected in the filamentous fungus P. chrysogenum nor in the
nonmethylotrophic yeasts S. cerevisiae and S. pombe (Fig. 8).
We have previously isolated an RNA-binding cellular protein,
Hv-p68, that copurifies with viral dsRNA from the filamentous fungus
C. victoriae, and demonstrated that it accumulates to higher levels in virus-infected isolates than in virus-free isolates (13). In
the present study, we isolated and completely sequenced a cDNA
containing the full-length ORF of the Hv-p68 gene.
Sequencing and phylogenetic analyses clearly indicated that Hv-p68
belongs to the family of FAD-dependent GMC oxidoreductases
(30) and that it is most closely related to the alcohol oxidases (AOX) of methylotrophic yeasts. Unlike the genes of the closely related AOX,
which are intronless, the Hv-p68 gene contains at least four introns. This finding has interesting evolutionary implications considering that Hv-p68, encoded by a gene from a filamentous fungus,
has significantly high sequence identity with this group of
flavoproteins from methylotrophic yeasts (sequence identities higher
than 67%). Hv-p68 gene homologues may have existed prior to
the divergence of yeasts and filamentous fungi. Furthermore, it is
possible that methylotrophic yeasts may harbor viruses that are
evolutionarily related to those infecting Cochliobolus species.
An increasing number of NAD+-dependent
dehydrogenases and oxidoreductases have recently been reported to
possess RNA binding activities (35-39). The dinucleotide-binding sites
of these enzymes share a typical The RNA binding activity of Hv-p68 has been demonstrated by gel
mobility shift experiments (13) and Northwestern blot analysis (this
study). Cellular proteins that bind to viral RNA may serve as
components of RDRP or may serve to bring various regions of a viral RNA
template together to form transcription or replication complexes (1,
40). Many viral RDRPs do not bind to viral RNA specifically or at all
(1). Considering that the RDRPs of the Hv145SV (encoded by a
monocistronic dsRNA)2 and of
the Hv190SV (9) are expressed independent of the CP, Hv-p68 may serve
to mediate the binding of RDRP to its template RNA during virion
assembly. The AOX of P. pastoris has also been shown to
exhibit limited RNA binding activity (this study). Its affinity for
viral transcripts, however, was significantly lower than that of
Hv-p68.
In addition to RNA binding activity, Hv-p68 also exhibits
phosphotransferase/kinase
activities.3 In this regard,
the NAD+-dependent glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) provides an excellent example for a comparable
multifunctional cellular protein with diverse biological properties.
GAPDH, once considered a simple classical glycolytic protein, is now
known to display a number of different activities, including nuclear
RNA export, DNA repair, translational control of gene expression, and
phosphotransferase/kinase activities (for a review, see Ref. 41).
Furthermore, several investigations suggest that GAPDH is involved in
apoptosis and viral pathogenesis (41).
The low methanol-oxidizing activity of the gradient purified Hv-p68
suggests that either methanol is not the natural substrate or that the
bulk of the protein in our purified preparations is present in an
inactive form. It is well accepted that FAD binding is a crucial step
in alcohol oxidase octamerization and that the FAD-containing octamer
comprises the active form of AOX (42). Our previous data have shown
that Hv-p68 occurs as an octamer in the Hv-p68-containing sucrose
gradient fraction, and thus it is presumed to be in its active form.
The release of FAD from Hv-p68 octamers during purification, however,
cannot be ruled out. It is more likely that methanol is not the natural
substrate for Hv-p68 as supported by the finding that Hv-p68 expression was neither induced by methanol nor suppressed by glucose when these
two supplements were used as the sole carbon source in cultures of
virus-free H. victoriae. It is noteworthy that, despite the high percentage of sequence identity in the coding region of AOX, the
5'- and 3'-untranslated regions show no significant similarities (33).
It is thus plausible that different transcriptional activators may
induce their expression. Comparison of Hv-p68 expression levels in
virus-infected and virus-free isolates (this study) clearly demonstrated that overexpression of Hv-p68 is associated with virus infection.
It may not be surprising that the bacterially expressed Hv-p68 was
completely inactive in the alcohol oxidase assay. Based on current
knowledge, the bacterially expressed Hv-p68 is neither expected to bind
FAD nor to oligomerize, and thus would lack oxidase activity.
Species-specific proteins (chaperons) are believed to be involved in
mediating FAD binding and oligomerization (42, 43). This is supported
by the finding that the AOX from H. polymorpha failed to
bind FAD and to oligomerize when synthesized in the heterologous host
S. cerevisiae (43). The absence of suitable chaperones in
the heterologous systems could explain these observations.
The finding that Hv-p68 is overexpressed in virus-infected fungal
isolates that exhibit the diseased phenotype (44) is of considerable
interest. We have shown by Northern hybridization analysis (this study)
that Hv-p68 mRNA levels were 10- to 20-fold higher in
virus-infected isolates than in virus-free isolates. Although the
identity of the natural substrate for the oxidase activity of Hv-p68 is
not known, the structurally similar alcohol oxidases from
methylotrophic yeasts or filamentous fungi mostly oxidize aliphatic
primary alcohols (and in a few cases, aromatic alcohols) irreversibly
to aldehydes, which are toxic (35, 45). A buildup of such toxic
intermediates when Hv-p68 is overproduced in virus-infected isolates
may lead to the lytic/diseased phenotype in virus-infected C. victoriae isolates (44).
Although we have only examined a limited number of fungal species, it
is apparent that all species in the genus Cochliobolus contain genes closely similar to Hv-p68. In a recent
phylogenetic study of the genus Cochliobolus (family
Pleosporaceae, order Pleosporales), it was determined that isolates of
C. victoriae are not monophylatic (46). This finding is in
agreement with our results on Southern analysis of restriction
fragments from three isolates of C. victoriae (Fig. 8),
which also showed that isolate 408 could be differentiated from other
isolates of C. victoriae. It will be of interest to determine the distribution of the Hv-p68 gene or homologues
in the fungal species belonging to the family Pleosporaceae or the order Pleosporales, which include several economically important plant
pathogens. Transcriptional activation of the Hv-p68 gene or
homologues leading to accumulation of toxic products, and hence a
diseased phenotype (as postulated for C. victoriae) could be exploited as a novel approach for biocontrol of such important plant pathogens.
-
-
fold motif. The Hv-p68 gene, or closely
similar genes, was present in all species of the genus
Cochliobolus but absent in the filamentous fungus,
Penicillium chrysogenum, as well as in two
nonmethylotrophic yeasts examined. This study represents the first
reported case that a member of the FAD-dependent glucose
methanol choline oxidoreductase family, Hv-p68, may function as an
RNA-binding protein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-
-
fold motif. In addition, we
demonstrate by Southern hybridization analysis that the
Hv-p68 gene, or closely similar genes, is present
in all Cochliobolus species examined but not in the
filamentous fungus Penicillium chrysogenum nor in two
nonmethylotrophic yeast species.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
0.5 °C per
cycle) for 2 min, 72 °C for 3 min, 30 cycles; 94 °C for 1 min,
50 °C for 2 min, 72 °C for 3 min, 10 cycles; 72 °C for 12 min). The PCR product generated (1.5 kbp) was gel-purified using
GeneClean (BIO 101) and blunt-end-cloned into an
EcoRV-digested pZErO vector (Invitrogen). Four clones were subjected to automated sequencing (ABI), and the sequencing information was used to design a sequence-specific primer (P3). A
750-bp fragment corresponding to the 3'-end of the Hv-p68 mRNA was
amplified from synthesized double-stranded cDNA by PCR using an
oligo(dT) NotI primer and P3, a primer based on Hv-p68
cDNA sequences at nucleotide (nt) positions 1396-1420 (see Fig.
1). Amplification reactions were carried out using Platinum High
Fidelity Taq DNA polymerase (Life Technologies, Inc.), and
the cycling parameters for TD-PCR were as specified above. The
amplified PCR product was gel-purified by GeneClean (BIO 101),
blunt-end-cloned into an EcoRV-digested pZErO vector
(Invitrogen), and subjected to automated sequencing (ABI).
-D-galactopyranoside and incubation for
a period of 5 h at 30 °C. Cells were collected by
centrifugation at 4000 × g for 5 min at 4 °C,
resuspended in 0.1 M sodium phosphate buffer (pH 7.2)
containing 0.1% Triton X-100, and treated with lysozyme (100 mg/ml
final concentration) for 15 min, at 30 °C followed by treatment with
DNase 1 (10 µg/ml final concentration) in 0.1 M sodium
phosphate buffer (pH 7.2) containing 10 mM
MgCl2 for 10 min at 30 °C. Bacterial extracts were
briefly sonicated and centrifuged at 14,000 × g for 15 min at 4 °C, and the pellet containing the inclusion bodies was
resuspended in 6 M guanidinium hydrochloride in binding
buffer (0.5 M NaCl, 20 mM Tris-HCl, pH 8.0).
The protein was solubilized by incubation on a rocker platform for
2 h at 4 °C. The His-tagged Hv-p68 was purified by Ni-NTA
chromatography, under denaturing conditions, as described by the
manufacturer (Novagen). The purified protein was renatured by
step-dialysis with successive changes of 50 mM Tris-HCl, pH
7.5, 50 mM NaCl buffer containing 6, 4, or 2 M
urea or no urea. The final protein preparation was concentrated using a
Centricon-10 device (Amicon).
-D-galactopyranoside and incubation for
a period of 4 h at 37 °C. Cells were collected by
centrifugation at 4000 × g for 10 min at 4 °C and
resuspended in 50 mM Tris, pH 7.5, 0.1% Triton X-100. The
bacterial extracts were sonicated and centrifuged at 14,000 × g for 15 min at 4 °C to separate the soluble and the
insoluble component (inclusion bodies). The overexpressed proteins were
purified from the inclusion bodies by repeated resuspension of the
pellet (in 50 mM Tris, pH 7.5, 0.1% Triton X-100) by brief sonication followed by centrifugation at 27,000 × g,
10 min at 4 °C. Five cycles of washing/centrifugation of the
resultant inclusion bodies pellet resulted in 90-95% removal of
contaminant proteins from the Hv-p68 preparation. The inclusion bodies
were finally resuspended in 500 µl of 50 mM Tris buffer,
pH 7.5, containing 50 mM NaCl and an equal volume of 2×
SDS-PAGE sample buffer, and the proteins were separated by SDS-PAGE for
Northwestern analysis.
-32P]dCTP-random-primed (Promega) Hv-p68 cDNA
probe at 42 °C for 16-18 h in 50% formamide hybridization solution
(50% formamide/1× Denhardt's solution/5× SSC/0.5% SDS/20
mM sodium phosphate, pH 7.0) and washed using high
stringency conditions (2× SSC, 0.2% SDS, 23 °C for 10 min; 0.2×
SSC, 0.1%SDS, 58-65 °C for 30-60 min). The Hv-p68 probe consisted
of a gel-purified, GeneClean (BIO 101)-extracted EcoRI-SstI restriction fragment (850 bp) from a
pZErO construct containing Hv-p68 cDNA. Genomic DNA was
isolated from filamentous fungal species by a modification of the
protocol described by Yoder (23). Mycelia from 200-ml shake cultures of
the following filamentous fungi were grown in a complete medium lacking
glucose for 4-5 days: H. (Cochliobolus)
victoriae, isolates A-9, B-2ss, and 408; C. heterostrophus; C. zeicola; C. sativum; and
P. chrysogenum. Mycelia were collected by straining through
two layers of Miracloth (Calbiochem), freeze-dried, and ground in
liquid nitrogen. The powdered mycelia were resuspended in 20 ml of
lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM
EDTA, 1% Sarkosyl) containing 6 mg of proteinase K (Life Technologies,
Inc.) and incubated at 50-55 °C for 60 min with occasional mixing
by inversion. The volume was increased to 50 ml with lysis buffer, and
the suspension was centrifuged at 10,000 × g for 10 min. The supernatant was extracted with Tris buffer-saturated phenol
(10 mM Tris-HCl, pH 7.5). The aqueous phase was
re-extracted by chloroform/isoamyl alcohol (24:1), ethanol-precipitated
(in 0.3 M sodium acetate), and the resultant pellet was
dried and resuspended in 2 ml of Tris-EDTA (TE) buffer. The DNA
preparation was treated with RNase A (20 µg/ml) at 37 °C for 20 min, the volume increased to 10 ml with TE buffer, and the DNA was
re-extracted with phenol/chloroform/isoamyl alcohol (25:24:1) followed
by extraction with chloroform/isoamyl alcohol (24:1). The DNA was
ethanol-precipitated in the presence of 0.3 M sodium
acetate, and the pellet was air-dried and resuspended in TE buffer. The
DNA was quantified by agarose-gel electrophoresis and
spectrophotometric analysis. For the isolation of genomic DNA from
yeast, 100-ml suspension cultures of S. cerevisiae and S. pombe were grown in YPD medium at 30 °C with shaking
to a density of 2 A600 nm. Cells were pelleted
by centrifugation at 3000 × g for 10 min, and the cell
pellet was resuspended in 2 ml of lysis buffer (2% Triton X-100, 1%
SDS, 100 mM NaCl, 1 mM EDTA, 10 mM
Tris-HCl, pH 8.0) and 2 ml of phenol/chloroform/isoamyl alcohol
(25:24:1). Three grams of acid-washed glass beads (450-500 µm,
Sigma) were added, and the suspension was vortexed intermittently, at
maximum speed, for 6-10 min. Two milliliters of TE buffer was added,
and the phases were separated by centrifugation at 6000 × g for 10 min. The aqueous phase was re-extracted with
chloroform/isoamyl alcohol (24:1) and ethanol-precipitated. The DNA
pellets were dried, resuspended in 1 ml of TE buffer, and treated with
50 µg of RNase A (at 37 °C for 15 min), and the DNA was
reprecipitated by addition of 100 µl of 4 M ammonium
acetate and 2.5 ml of ethanol. The DNA was resuspended in TE buffer and
quantified by agarose-gel electrophoresis and spectrophotometry.
-32P]dCTP-random-primed (Promega) Hv-p68 cDNA
probe for 16-18 h at 42 °C in 50%-formamide hybridization solution
and washed using high stringency conditions (2× SSC, 0.2% SDS, at
23 °C for 10 min; 0.2× SSC, 0.1% SDS, at 58-65 °C for 30-60
min). The Hv-p68 probe consisted of a gel-purified, GeneClean (BIO
101)-extracted, EcoRI-SstI restriction fragment
(850 bp) from a pZErO construct containing a cDNA of
Hv-p68.
A405 nm/min (units) per mg of protein.
-32P]UTP. The
transcription reactions were terminated by treatment with RQ1
RNase-Free DNase (2 units, for 15 min at 37 °C; Promega), and the
reaction mixtures were extracted with phenol/chloroform, ethanol-precipitated, and resuspended in diethyl
pyrocarbonate-treated water. The specific activity of the
riboprobes was determined by scintillation counting of the
trichloroacetic acid-precipitable material.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide and deduced amino acid sequence of
Hv-p68 cDNA. The coding region, 1998 nt, including the stop
codon (boxed) at nt positions 1996-1998, codes for a
polypeptide of 665 amino acid residues with a predicted size of 74,251 Da. Potential sites for N-linked glycosylation of the
conserved sequence NX(S/T) are underlined.
Peptides whose sequences were determined from purified Hv-p68 and which
were used to design degenerate oligonucleotide pools (P1 and
P2) are printed in boldface (arrows
indicate the direction of primers). P3 (nt sequence in
boldface) corresponds to the gene-specific primer used along
with primer oligo(dT)-NotI to direct the amplification of a
750-bp 3'-end fragment of Hv-p68 by PCR. The tripeptide SRL
(shaded box) located at the C terminus corresponds to the
conserved signal, PTS1, for peroxisomal translocation.
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Fig. 2.
Nucleotide sequence of a partial genomic
clone of Hv-p68 and positions of four introns (I-IV). The nt
sequence of a partial genomic clone (1723 bp) was compared with a
corresponding Hv-p68 cDNA clone (1511 bp). Transcribed nts in the
genomic sequence are shown by uppercase letters
(corresponding nts in the cDNA sequence are indicated by
dots), whereas intron sequences are in lowercase
letters. The putative 5'- and 3'-splice sites are printed in
boldface; the consensus internal sites are
underlined.
-
-
fold motif (Fig. 3; see also Ref. 31). The three Gly
residues between the first
-sheet and
-helix as well as the
acidic amino acid at the end of the second
-sheet are absolutely
conserved in these flavoenzymes (Fig. 3; printed in
boldface), as well as in other homologous oxidoreductases
(32, 33). Although the functions of the other signature patterns (blocks B-E) are not yet definitively known, their putative functions have been discussed in a recent study (34). Block B comprises the
flavin attachment loop (34). A conserved region near the C terminus
(Fig. 3, boxed) that is shared by GMC oxidoreductases (33,
35) is believed to correspond to the active site. Differences in
substrate specificity of alcohol oxidases (aliphatic or aromatic alcohols) are probably due to differences in the active site of these
enzymes (35).
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Fig. 3.
A multiple sequence alignment of the deduced
amino acid sequence of Hv-p68 and four alcohol oxidases from
methylotrophic yeasts. The alignment was generated by the PILEUP
program, and the consensus was produced using the PRETTY program. The
amino acids comprising the five signature blocks, A-E,
typical of GMC flavoproteins are shaded. The position of the
conserved FAD ADP-binding region (a typical -
-
fold motif)
near the N terminus is indicated by a horizontal line. The
three glycine residues in the GXGXXG motif
between the first
-sheet and
-helix and also the acidic
amino acid, glutamic acid (E), at the end of the second
-sheet are absolutely conserved in these flavoproteins. The
conserved region near the C terminus, corresponding to the active site,
is boxed. The C-terminal tripeptide with consensus sequence
for the peroxisomal translocation signal, PTS1, is
shaded.
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Fig. 4.
Pairwise sequence comparison and phylogenetic
analysis of Hv-p68 and representative members of the
FAD-dependent GMC oxidoreductases. A,
percentage of deduced amino acid sequence similarity (identity) between
Hv-p68 and representative members of the family of
FAD-dependent GMC oxidoreductases. Values from paired
alignments were generated by the GAP program. Percentages of
similarity/identity are given above/below the diagonal
line, respectively. B, phylogenetic relationships
between Hv-p68 and members of the FAD-dependent GMC
oxidoreductase family. Tree distances were calculated using the
Neighbor-Joining method from an alignment generated by ClustalW, and
the resulting tree was displayed by TreeView. The consensus tree was
supported by the analysis of 1000 bootstrap replicates (bootstrap
values are given at branch nodes). AOX1-Pp (GenBankTM accession number
U96967-1) and AOX2-Pp (accession number U96968-1) alcohol oxidases of
P. pastoris; AOX-Pa alcohol oxidase (accession number
A11156-1) of P. angusta; MOX-Cb (accession number Q00922)
alcohol oxidase of Candida boidinii; AOX1-Pm (accession
number AAF02494) and AOX2-Pm (accession number AAF02495) alcohol
oxidases of P. methanolica; GOX-An (accession number A35459)
glucose oxidase of Aspergillus niger; and GLD-Ds (accession
number AAB87896) glucose dehydrogenase of Drosophila
subobscura. GOX-An was included as an outgroup to determine the
proper root placement.
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Fig. 5.
Immunological reactivity and RNA binding
activity of bacterially expressed Hv-p68. A, an Hv-p68
cDNA containing the full-length coding region was introduced into
the bacterial expression vector pET22(b)+ to generate construct pETp68,
and the overexpressed protein was extracted and purified by Ni-NTA
chromatography. Hv-p68 purified from fungal extracts (Hv-p68
lane), the Ni-NTA-purified bacterially expressed protein
(NTA-pET-p68 lane), and alcohol oxidase from P. pastoris (Pp-AOX lane) were analyzed by Western
blotting using antibodies to Hv-p68. The Hv-p68 antiserum reacted
strongly with its homologous antigen (Hv-p68) as well as with the
bacterially expressed protein (NTA-purified pETp68), but its reaction
with Pp-AOX was comparatively weaker. B, RNA binding
activities of purified fungal Hv-p68, NTA-purified pET-p68, and Pp-AOX
were tested by Northwestern blotting analysis using
32P-labeled in vitro transcripts of cloned
cDNAs to Hv190SV dsRNA and Hv145S dsRNA-4 (riboprobes). Native and
bacterially expressed Hv-p68 showed strong binding to both probes.
Binding of Pp-AOX to either probe was very weak, detectable only in
overexposed autoradiographs.
-
-
dinucleotide binding fold (Rossman fold). It has been proposed that the
dinucleotide-binding sites and the RNA-binding domains are structurally
related (36). It was of interest, therefore, to determine whether the
FAD ADP-binding domain in Hv-p68 might also function as an RNA-binding
domain. To localize the RNA-binding domain, we expressed N- and
C-terminal truncations of the Hv-p68 protein in E. coli and
compared their RNA-binding activities by Northwestern blot
analysis. The bacterially expressed proteins were purified from
the inclusion bodies as single major protein bands with molecular
masses of ~54 and 62 kDa for the N- and C-terminal deletions,
respectively (Coomassie-stained gel; Fig.
6). In Northwestern blots, binding of
either of the RNA probes was similar for both the full-length and the
C-terminally truncated Hv-p68. On the other hand, deletion
of the N-terminal region of Hv-p68 completely abolished all binding to
the RNA probe (Fig. 6). These results indicate that the RNA-binding
domain is localized within the N-terminal region of
Hv-p68.
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Fig. 6.
Comparative binding activities of bacterially
expressed full-length Hv-p68 and N- and C-terminal truncations.
Binding activity to radiolabeled transcripts was determined by
Northwestern analysis. Coomassie Blue-stained SDS-PAGE gel with
bacterially expressed Hv-p68 purified from inclusion bodies; N- and
C-terminally truncated Hv-p68 (estimated sizes of 54 and 62 kDa,
respectively). Similar RNA binding was observed for full-length and
C-terminally truncated HV-p68. The N-terminally truncated
Hv-p68 did not show any detectable binding to either radiolabeled
transcript (arrow and dotted line across
autoradiographs indicate position of the N-terminally truncated
Hv-p68). Northwestern assays were as described for Fig. 5.
Enzymatic assay for alcohol oxidase activity
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Fig. 7.
Northern analysis of Hv-p68 mRNA
transcript levels. A, total RNA (25 µg) isolated from
the virus-free isolates 408 and B-2ss and the virus-infected isolate
A-9 were electrophoresed on a formaldehyde/agarose gel, blotted, and
hybridized under high stringency conditions with a radiolabeled probe
for Hv-p68. mRNA, ~2.25 kb in size, was detected in RNA samples
from all three H. victoriae isolates. The level of Hv-p68
transcript in cultures of the virus-infected isolate A-9, however, was
at least 10-fold higher than that for the virus-free isolates.
B, total RNA (30 µg) isolated from cultures of the
virus-free isolate B-2ss grown for 3 days in minimal medium
supplemented with either glucose or methanol. Similar amounts of Hv-p68
mRNA were detected in total RNA isolated from fungal cultures
supplemented with either glucose or methanol, as a carbon source. The
32P-labeled Hv-p68-specific probe was generated by
random-primer labeling of a 850-bp EcoRI-SstI
restriction fragment from cloned Hv-p68 cDNA.
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Fig. 8.
Southern analysis of genomic DNA from several
Cochliobolus species, P. chrysogenum,
and nonmethylotrophic yeasts. Genomic DNA was digested with
restriction enzymes (as indicated) and resolved on a 0.8%
agarose gel. The blotted DNA restriction fragments were
hybridized under high stringency conditions with a
32P-labeled probe generated by random-primer labeling of a
850-bp EcoRI-SstI restriction fragment from
cloned Hv-p68 cDNA (nt positions 41-850 in Fig. 1). Fungal species
used: Cv, C. (Helminthosporium)
victoriae; Ch, C. heterostrophus;
Cz, C. zeicola; Cs, C. sativus; Pc, P. chrysogenum; Sc,
S. cerevisiae; Sp, S. pombe.
Restriction enzymes used: p, PstI; X,
XhoI; B, BglI; b,
BamHI; E, EcoRI and
EcoRV.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-
-
fold motif, which is
proposed to comprise the RNA-binding domain. The finding that RNA
binding activity of Hv-p68 is localized at the N-terminal region
containing the putative ADP-binding domain presents the first reported
example of an FAD-dependent oxidoreductase that may
function as an RNA-binding protein.
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FOOTNOTES |
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* This work was supported by Grant Agreement 96-35303-3240 from the United States Department of Agriculture National Research Initiative Competitive Grants Program (to S. A. G.) and is published with the approval of the Director of the Kentucky Agricultural Experiment Station as Journal Article 00-12-139.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) AF232903.
To whom correspondence should be addressed: Dept. of Plant
Pathology, University of Kentucky, S-305 Agricultural Sciences Center-N, Lexington, KY 40546-0091. Tel.: 859-257-5969; Fax:
859-323-1961; E-mail: saghab00@pop.uky.edu.
Published, JBC Papers in Press, October 30, 2000, DOI 10.1074/jbc.M007701200
2 A. I. Soldevila, W. M. Havens, S. Huang, and S. A. Ghabrial, manuscript in preparation.
3 S. A. Ghabrial and W. M. Havens, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: dsRNA, double-stranded RNA; PAGE, polyacrylamide gel electrophoresis; FAD, flavin adenine dinucleotide; GMC, glucose methanol choline; TD-PCR, touchdown-polymerase chain reaction; DOP-PCR, degenerate oligonucleotide primer-polymerase chain reaction; AOX, alcohol oxidase; MOX, methanol oxidase; bp, base pair(s); kbp, kilobase pair(s); Hv, Helminthosporium (Cochliobolus) victoriae; ORF, open reading frame; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PTS, peroxisomal targeting signal(s); NAD, nicotinamide adenine dinucleotide; Ni-NTA, nickel-nitrilo-triacetic acid resin; CP, capsid protein; RDRP, RNA-dependent RNA polymerase; nt, nucleotide(s).
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