Content uploaded by Rune Hartmann
Author content
All content in this area was uploaded by Rune Hartmann on Jan 15, 2014
Content may be subject to copyright.
1998 Oxford University Press
4121–4127
Nucleic Acids Research, 1998, Vol. 26, No. 18
p59OASL, a 2′–5′ oligoadenylate synthetase like protein:
a novel human gene related to the 2′–5′ oligoadenylate
synthetase family
Rune Hartmann, Henrik Steen Olsen, Stefanie Widder, René Jørgensen and Just Justesen*
Department of Molecular and Structural Biology, University of Aarhus, C. F. Møllers allé Building 130, DK-8000 Aarhus C,
Denmark and
1
Human Genome Sciences Inc., 9410 Key West Avenue, Rockville, MD 20850, USA
Received June 22, 1998; Revised and Accepted July 29, 1998 DDBJ/EMBL/GenBank accession nos AJ225089 and AJ225090
ABSTRACT
The 2′–5′ oligoadenylate synthetases form a well con-
served family of interferon induced proteins, presumably
present throughout the mammalian class. Using the
Expressed Sequence Tag databases, we have identified
a novel member of this family. This protein, which we
named p59 2′–5′ oligoadenylate synthetase-like protein
(p59OASL), shares a highly conserved N-terminal
domain with the known forms of 2′–5′ oligoadenylate
synthetases, but differs completely in its C-terminal
part. The C-terminus of p59OASL is formed of two
domains of ubiquitin-like sequences. Here we present
the characterisation of a full-length cDNA clone, the
genomic sequence and the expression pattern of this
gene. We have addressed the evolution of the 2′–5′
oligoadenylate synthetase gene family, in the light of
both this new member and new 2′–5′ oligoadenylate
synthetase sequence data from other species, which
have recently appeared in the databases.
INTRODUCTION
The 2′–5′ oligoadenylate (2–5A) system is a regulated RNA
decay pathway, consisting of a number of 2′–5′ oligoadenylate
synthetases (2–5A synthetases) and a 2–5A activated ribonuclease,
normally referred to as RNase L. The 2–5As were originally
described as low molecular weight inhibitors of protein synthesis,
which were produced in cell free extracts of interferon treated
cells, especially following incubation with Poly I·Poly C (1,2).
The ability of 2–5As to bind to and activate RNase L, resulting
in a general RNA degradation, makes them potent inhibitors of
protein synthesis (3,4).
The 2–5As are produced by a family of enzymes, the 2′–5′
oligoadenylate synthetases (2-5A synthetase or OAS, EC 2.7.7.-).
Hitherto, two human genes encoding highly homologous 2–5A
synthetases have been described, referred to as the p42OAS gene
and the p69OAS gene. Both genes are situated on chromosome
12 (5). The p42OAS gene encodes two splice variants, which
differ only at their C-termini, having a molecular weight of 42 and
46 kDa (6,7). Also the p69OAS gene encodes two splice variants
differing at their C-termini, having theoretical molecular weights
of 79 and 83 kDa, but migrating with apparent molecular weights
of 69 and 71 kDa, respectively (8). Most likely, the p69OAS gene
arose from a duplication of an ancestral 2–5A synthetase gene, since
it harbours two repeated regions, each with ~40% homology to the
p42OAS form (8). Furthermore, immunological evidence exists for
a 2–5A synthetase with an apparent molecular weight of 100 kDa
(9), which has also been found by purification procedures (10).
The biosynthesis of all the known 2–5A synthetases is
stimulated by interferon (reviewed in 11). 2–5A synthetase is
produced as a latent enzyme which is activated by certain classes
of RNA, mainly double stranded RNA (dsRNA) (12). Several
studies have dealt with stimulation of semipurified 2–5A
synthetases by Poly I·Poly C and analogues thereof (reviewed in
13). Recently, we addressed the structural requirements of the
RNA activator of pure 2–5A synthetase (p46 isoform) (12).
The involvement of the 2–5A system in the interferon induced
antiviral effect has been inferred from several sets of experiments.
For instance, over-expression of the p42OAS in Chinese hamster
ovary (CHO) cells gives resistance to infection by picornavirus,
but not to vesicular stomatitis virus (VSV) (14). In addition,
transgenic plants expressing 2–5A synthetase as well as RNase L
show a remarkable increase in their resistance to viral infections
(15,16). The 2–5A system has also been suggested to play a role
in the growth suppression engendered by interferons (17).
Expression of a dominant negative RNAse L mutant in murine
cells resulted in suppression of both the interferon mediated
protection against picornavirus and the antiproliferative effect
otherwise exerted by interferon (18). Recent evidence from
RNase L deficient mice strongly suggests that apoptosis can be
induced via the 2–5A system. The RNase L –/– mice had an
enlarged thymus, caused by the lack of normal apoptosis of
thymocytes and fibroblasts in the thymus (19).
In general, RNase L is expressed in all tissues and only weakly
induced by interferon, (reviewed in 20), therefore the synthesis of
2–5As seems to be the controlling step in inducing RNA decay
by the 2–5A pathway. The different isoforms of 2–5A synthetase
apparently have similar enzymatic capacities, but the isoforms
seem to have different tissue specific expression patterns and to
respond differently to treatment with various cytokines including
interferons (reviewed in 11).
The recent development of Expressed Sequence Tag (EST)
databases and the increasing complexity of these databases
*To whom correspondence should be addressed. Tel: +45 89422682; Fax: +45 89422637; Email: jj@mbio.aau.dk
by guest on June 1, 2013http://nar.oxfordjournals.org/Downloaded from
Nucleic Acids Research, 1998, Vol. 26, No. 18
4122
(reviewed in 21) gives an opportunity for the identification of new
members of a given protein family. We therefore searched the
EST database for sequences displaying homology to 2–5A
synthetase. In the following, we thus present the cloning and
characterisation of a novel member of the human 2–5A synthetase
family and the corresponding gene.
MATERIALS AND METHODS
Cloning and sequencing
An EST clone (HG# 1173451) encoding a putative novel 2–5A
syntethase was identified in an EST database by searching with
the known human 2–5A family members P69OAS (M87284) and
p42OAS (P00973) using the BLAST search algorithm (22). This
clone was obtained from Human Genome Sciences and sequenced
in its full length by the primer walking approach. Sequencing was
performed on an Applied Biosystems 373A sequencer, using the
ABI Prism Dye Terminator Cycle Sequencing kit (Perkin Elmer).
Each strand was sequenced twice.
Rapid amplification of cDNA ends (RACE)
The RACE was performed as described in (23). Total RNA from
HeLa cells was used as the RNA source. The primers for the
second nested PCR contained a BamHI/XhoI restriction site,
respectively, which allowed cloning of the resulting PCR product
in the BamHI/XhoI sites of the pBluescript SK vector (Stratagene).
The clones were sequenced using the general sequencing primers
M13-21 and M13 reverse (DNA technology, Aarhus, Denmark).
Primer sequences were as follows:
Gene Specific Primer 1 (GSP1) TCCATATCAGCCTCAGAAC;
GSP 2 (including a BamHI site) CGCGGATCCGGAAGCTGT-
GGAAACAGCTC; Linker primer 1 CAATCAAGAATCCCTG-
CTCAGCGTAA; and Linker primer 2 (including a XhoI site)
CGCCCTCGAGCCTCAACACCTACCCTATC.
Alignment
The alignment was established using the following sequences:
Human P69OAS (M87284), Human p59OASL (AJ225089),
Human p42OAS (Swissprot P00973), Mouse OAS (L3)
(M33863), Rat OAS (Z18877), Porcine OAS (AJ225090) and
Chicken OAS (AB002585) (accession numbers in parentheses).
Alignment was performed with the clustalw programme (clustalw,
an interactive service at the http://www2.ebi.ac.uk/clustalw ) using
default parameters (24). The alignment was resistant to changes of
parameters. The output was embellished using the BOXSHADE
software (http://ulrec3.unil.ch/software/BOX_form.html ).
Construction of phylogenetic trees
The phylogenetic tree was constructed using the software
supplied with the Phylip package (version 3.57) (25). The tree
shown was created using maximum parsimony analysis on multiple
aligned sequences (program PROTPARS, default parameters). The
bootstrap re-sampling method was used (100 replicates) to assess
the confidence of each node in the maximum parsimoni tree
(programs SEQBOOT and CONSENSE). Phylogenetic trees
constructed using the neighbour-joining algorithm on the same
set of data gave a congruent phylogeny [programs PROTDIST
(setting DAYHOFF matrix) and NEIGHBOR (default parameters),
or using Maximum Likelihood analysis (program PROTML,
default settings)].
Northern blotting
Northern blots were performed using MTN filters from Clontech.
The filters were hybridised in Hybrisol (Oncor) overnight at
42C, at a probe concentration of ∼1.5 × 10
6
c.p.m./ml. After
hybridisation the blots were washed at high stringency at 65C
and exposed to a film. The probe was prepared by isolating a
BamHI/XhoI fragment of the p59 coding sequence (nucleotides
374–919) or a 1.3 kb EcoRI fragment from the p42 cDNA
(nucleotides 1–1275), from an agarose gel (Qiagen Gel Extraction
kit). The isolated fragment was radioactively labelled using
Stratagene Prime-it II kit and [α-
32
P]dCTP.
RNase protection assay
A XhoI/KpnI fragment of the p59 cDNA (280 bp) was cloned in
to the appropriate sites in the pBluescript SK(+) vector. After
linearisation with XhoI, T
7
transcription was performed according to
standard protocols using [α-
32
P]UTP as incorporated label. The
specific activity of the probe was ∼1 × 10
20
c.p.m./mol. The
γ-actin probe was prepared as described by Gunning et al. (26).
RNase protection was performed using the Ambion RPA II kit
(Cat. #1410), following the manufacturer’s instruction. p59 probe
(3 × 10
–16
mol) and 1 × 10
–15
mol of the γ-actin probe was
hybridised to 10 µg of total RNA from amniotic fluid (AMA)
cells, treated as indicated.
Cell culturing
AMA cells (gift from J. E. Celis, Aarhus, Denmark) were grown
in glutamax medium containing 10% newborn calf serum and
penicillin/streptomycin, induced with 500 U/ml interferon-α or
100 U/ml interferon-γ for the indicated time. RNA samples were
prepared by the acid guanidinium thiocyanate/phenol/chloroform
extraction method (27).
RESULTS AND DISCUSSION
EST identification and sequencing
We searched the EST database for clones showing similarity to
already known forms of 2–5A synthetase as described by Adams
et al. (28). An EST clone appeared that revealed homology to the
known forms of 2–5A synthetase and this clone was sequenced
in its full length. The clone displayed a long open reading frame
(ORF) and the corresponding amino acid sequence showed
strong homology to known 2–5A synthetase proteins (Figs 1 and
2). The ORF had no obvious start codon, so we assumed that the
clone was truncated at its 5′ end.
To obtain the missing 5′ part of the cDNA clone we performed
the new RACE procedure on total RNA from HeLa cells (Fig. 3).
The resulting PCR fragment was extracted from the gel and
cloned in the BamHI/XhoI sites of the pBluescript SK vector. Ten
individual clones were sequenced of which six contained an
additional 19 bp, compared with the original EST sequence. The
other three clones were truncated in a manner similar to the
original EST clone and one contained no insert. Since no longer
bands were observed in the PCR, even after extending the
elongation time to 4 min, we assume that the band observed
represents the authentic 5′ end.
by guest on June 1, 2013http://nar.oxfordjournals.org/Downloaded from
4123
Nucleic Acids Research, 1994, Vol. 22, No. 1
Nucleic Acids Research, 1998, Vol. 26, No. 18 4123
The additional 19 bp that were found by ‘new RACE’
contained two in frame start codons. As the two AUG codons are
only separated by 6 nt it is possible that either one could be used
as an initiation codon (29). In this case, however, the second AUG
codon is placed in a context making it highly unlikely to be used
as the principal start codon (30) (the normal adenine in position
–3 and the normal guanine in position +4 are both substituted by
a cytosine). The complete cDNA clone contains 1819 bp and has
one ORF coding for a 514 amino acid protein with a predicted
molecular mass of 59 226 Da, hence called p59OASL (59 kDa
2–5 oligoadenylate synthetase-like protein) (accession number
AJ225090). The size of the cDNA agrees well with the observed
size of the messenger (1.8 kb) in northern blots (Fig. 4).
A 3′ fragment of the p59 gene has previously been described as
a thyroid receptor interactor (TRIP14) due to a yeast two-hybrid
screening (31), where a part of p59 (amino acids 260–413) is
reported (Q15646). We have not investigated the possible link of
p59OASL to the thyroid hormone system.
Gene structure
Using the cDNA sequence as input we searched the High
Throughput Genomic Sequences databases using the BLAST
algorithm. We found a clear match to a bac-clone 92N15 currently
under sequencing at the Sanger center (Cambridge, UK). This
clone is part of the MODY3 (Maturity onset of diabetes 3) region,
which maps to chromosome 12q24.2 (32). The highly homologous
genes p42OAS and p69OAS genes (5) also map to chromosome
12 where they link to the marker d12s1718 (Unigene Hs.82396
and Hs.24815, NCBI) with the genetic map position 129 cM and
Radiation Hybrid map position 576 cR. The bac-clone 92N15
contains the marker d12s2088 (R. Cox, personal communication),
so the p59OASL gene maps in the MODY3 region at map
positions 138 cM and 580 cR. The two markers d12s1718 and
d12s2088 are both contained in the WC12.8 contig (singly linked)
(Whitehead Institute, MA, USA).
Based upon the sequence data of bac-clone 92N15 from the
Sanger centre, we determined the structure of the p59OASL gene
to be composed of 6 exons. Exons 1–5 encode the 349 amino acid
N-terminal domain that has high homology to other 2–5A
synthetases, exon 6 encodes a 165 amino acid C-terminal domain
that has no homology to any of the known 2–5A synthetases
(Fig. 1A) but homology to two consecutive ubiquitins (see
below). Exon 6 also harbours a putative poly A signal.
The gene structure of the p42OAS gene has previously been
determined (6). This gene is composed of 7 exons with the
translational start site situated in exon 3. By comparing the intron
exon boundaries of the p59OASL gene to the p42OAS gene, it
became evident that exons 3–7 of the p42OAS gene have a structure
similar to that of exons 1–5 of the p59OASL gene (Fig. 1B). This
suggests that the two genes have arisen from a duplication of an
ancestral gene; exon 6 of p59OASL has thus been fused to the
duplicated gene by exon shuffling. The two untranslated exons of
the p42OAS gene have either been lost in the p59OASL gene or
added to the p42OAS gene after it diverged from p59OASL.
Both the gene structure of p59OASL and the primary structure
of the p59OASL protein suggest a two domain structure of the
p59OASL protein, consisting of an N-terminal 2–5A synthetase
domain of ∼350 amino acids encoded by exons 1–5 and a
C-terminal domain on 165 amino acids encoded by exon 6.
Figure 1. (A) Organisation of the p59OASL gene. Upper numbers refer to the
first and last nucleotide in an exon, according to the cDNA for p59OASL.
Numbers in the lower position indicate the size of a given intron. (B) The amino
acid sequence of the p42OAS and p59OASL proteins were aligned using the
clustalw program. The first and last amino acid in each exon is highlighted. The
single highlighted leucine indicates the possible alternative splice site in the p42
gene, which is used to give the p46 variant.
The promoter region 3000 bases upstream of the initiation site
was analysed for the presence of transcription factor binding sites.
Among the numerous putative sites several putative ISRE sites
around –150, –290, –330 and –960 nt can be linked to classical
interferon induction pathways including STAT1 and IRF1 (33).
However, we have also found one putative IRF1 and two IRF2
sites in the middle of the ∼10 kb intron 1, which might explain the
moderate interferon induction detected in the RNase protection
assays (see later). However, the activity of these sites has to be
determined.
Primary structure of the p59OASL protein
We aligned the amino acid sequence of seven different 2–5A
synthetases, available in the Swissprot or GenBank databases
(Fig. 2). As previously mentioned, the human p69OAS is
composed of a repeat of two domains each showing homology to
the p42OAS. Therefore, the p69OAS gene is represented by two
sequences in the alignment p69N and p69C, corresponding to the
N-terminal and C-terminal domains, respectively. Since the
C-termini of various 2–5A synthetase isoforms are poorly
conserved, and apparently not important for enzymatic function
(34), we have only applied sequences N-terminal to the CFK
motif (amino acids 331–333, p42OAS numbers), recently shown
to be essential for enzymatic activity (35).
Recent mutational studies showed that a LXXXP motif at the
N-terminus is important for enzymatic function. If either the
by guest on June 1, 2013http://nar.oxfordjournals.org/Downloaded from
Nucleic Acids Research, 1998, Vol. 26, No. 18
4124
Figure 2. (A) Alignment of various 2–5A synthetases. The amino acid sequences of the N-terminal part of the indicated 2–5A synthetase proteins (see text), were
aligned using the clustalw program. (B) Alignment of the C-terminal part (165 amino acids) of p59OASL coded by exon 6, with ISG15, Human Ubiquitin-like protein
GDX (UBIL) and two consecutive ubiquitins separated by XXX. The aligned sequences were subsequently embellished using the BOXHADE program.
A
B
by guest on June 1, 2013http://nar.oxfordjournals.org/Downloaded from
4125
Nucleic Acids Research, 1994, Vol. 22, No. 1
Nucleic Acids Research, 1998, Vol. 26, No. 18 4125
Figure 3. PCR products from the RACE procedure were separated on a 1%
agarose gel. A strong band appears when the RNA was decapped prior to
ligation, but not if decapping was omitted. Prolonging the extension time to
4 min did not lead to new larger bands. Lane 1, PCR using decapped RNA and
an extension time of 1 min; lane 2, decapped RNA and a extension time of
4 min; lane 3, RNA not decapped prior to ligation, extension time 1 min; lane 4,
same as lane 3 but the extension time was 4 min.
Figure 4. Northern blots. Filters containing 2 µg of poly A
+
RNA pr. lane from
various tissues were probed with (A) a p59OASL specific probe or (B) a
p42OAS/p46OAS specific probe. Lane 1, spleen; lane 2, thymus; lane 3,
prostate; lane 4, testis; lane 5, uterus; lane 6, small intestine; lane 7, colon
(mucosal lining); lane 8, peripheral blood leucocytes. The p42/p46 probe gives
distinct bands corresponding to the p42 and p46 isoforms. The origin of the
larger bands is unknown.
leucine or proline were mutated, the enzyme would lose its
capability to form 2–5As but would still be able to bind both ATP
and dsRNA (36). This motif is conserved except in the chicken
2–5A synthetase form.
Generally the amino acids from 190 to 320 are well-conserved
(numbers according to the p59OASL sequence). Mutational and
biochemical studies placed the high affinity ATP binding domain
of p42OAS in the proximity of lysine 199 (p42 numbering) (37).
A stretch of ∼25 amino acids around lysine 199 is highly
conserved, except for some relaxation in the p69N sequence. We
also point out two other highly conserved motifs, the
YALTLLT(v/i)YAWE motif and the RP(v/i)ILDPADPT motif.
No functional data exist for those motifs.
Finally, there is a P-loop motif, presumably involved in
phosphate binding, in the p42OAS family which is especially
homologous to adenylate kinases (myokinases): VVXGXXSG-
KGT. The last glycine (G) is unique in the P-loop of these two
protein families compared to all other P-loops (38). This is a
striking phenomenon considering that the substrates for these two
very different enzymes actually are rather similar, ATP + ATP
versus ATP + AMP. However, in p59OASL the crucial lysine (K)
is replaced by an asparagine (N) which could be an indication of
the essential differences in the activity of p59OASL compared to
all the other 2–5A synthetases, including the most homologous
one, chicken 2–5A synthetase. The same region of the p42OAS
was proposed to share structural homologies to the rat DNA
polymerase β phosphate binding loop (39). The aspartate (D)
residues 75 and 77 are presumed to co-ordinate an active site
magnesium ion. In p59OASL these aspartates are replaced by
glutamates by single nucleotide substitutions. Glutamate remains
capable of co-ordinating the crucial magnesium ion, but the
change from aspartate to glutamate might lead to a difference in
substrate specificity or in the reaction conditions required.
Further mutational studies are necessary to shed light on the
functional role of the P-loop region of p59OASL, as well as the
other members of the 2–5A synthetase family.
The C-terminal domain, exon 6, of p59OASL shows homology
to the ubiquitin like proteins ISG15 [M13755, (18)] (identities =
40/165) and the ubiquitin-like protein GDX [P11441, (40)]
(identities = 31/165) (Fig. 2). All three proteins seem to have
evolved from a duplication of a ubiquitin gene. The C-terminus
of p59OASL_exon 6 is 36% homologous to ubiquitin [P02248,
(41)] (identities = 26/76) and the N-terminus of p59OASL_exon
6 is 23% homologous to ubiquitin (identities = 22/76). There are
examples of other proteins, ribosomal protein S30 (42) and large
proline rich protein BAT3 (43), which contain intrinsic ubiquitin
domains playing a role in the regulation of the activities and
half-lives of these proteins. It is thus tempting to speculate that the
C-terminal domain is involved in the regulation of either the 2–5A
synthetase activity or the stability of the protein. One might even
propose that a proteasome pathway is involved in a degradation
process leading to smaller forms of p59OASL, and that a
proteasomal activity is part of the post-translational control mechan-
ism of the activity of the p59OASL protein.
Phylogenetic studies
Using the alignments presented above, we have constructed a
hypothetical phylogenetic tree (Fig. 5), as described in Materials
and Methods. The p42OAS forms from four different species
(Rat, Mouse, Pig and Man) group in the same family (bootstrap
by guest on June 1, 2013http://nar.oxfordjournals.org/Downloaded from
Nucleic Acids Research, 1998, Vol. 26, No. 18
4126
Figure 5. Phylogenetic tree for the different 2–5A synthetase forms. Bootstrap
values are shown.
value 100%) whereas p59OASL groups together with a chicken
form of 2–5A synthetase (bootstrap value 95%). The two
sequences representing the p69OAS form either have a node
between them (as on the tree shown) or they group to their own
branch of the tree. Except for the slight variations in the placing
of the p69OAS sequences, the use of different algorithms and
change of parameters did not change the topology of the
phylogenetic tree.
From the topology of the phylogenetic tree it is evident that the
multiple isoforms seen in humans diverged before the radiation
of the mammalian class, meaning that a similar number of
isoforms are very likely to exist in other mammalian species.
The expression pattern of p59OASL
To evaluate the expression of the p59OASL messenger RNA in
different tissues we made northern blots using Multiple Tissue
Northern from Clontech (Fig. 4). A wide variety of tissues were
screened for p59OASL expression by this method. Low expression
could be detected in most tissues, but high expression was
detected in primary blood leukocytes (and other tissue related to
the haematopoetic system), in colon, stomach and to some extent in
testis, whereas the expression in small intestine was low (Table 1).
An aspect common to tissues with high expression of p59OASL
mRNA might be that they contain highly proliferating cells. The
cells of the lymphoid system as well as the cells present in colon
or stomach epithelium are rather short lived and are constantly
replaced.
To our knowledge, the expression patterns of the p42OAS or
p69OAS isoforms have not been addressed in detail, but
re-probing of the filter with a p42/p46 specific probe showed a
more widespread expression of this isoform (Fig. 4) compared to
p59OASL.
We investigated the induction of p59OASL by interferon-α and
interferon-γ in AMA cells. The transcription of p59 is stimulated
2–5-fold by the addition of interferon-α and 2–3-fold by
interferon-γ, as demonstrated by RNase protection assays (Fig. 6).
Using RT–PCR we have seen a similar induction in HeLa cells
(data not shown).
The data presented here suggest that the p59OASL expression
is strictly controlled, and only expressed in certain tissues.
Presumably this regulation correlates with the cellular function of
p59OASL. We are currently investigating whether p59OASL is
linked to the interferon system in a similar manner as the p42OAS
and p69OAS isoforms, or whether p59OASL is part of other
cytokine systems. In the view of the prevalence of p59OASL in
highly proliferating cells, we believe that a role may exist for
p59OASL as part of a general growth control pathway in those cells.
Table 1. Expression of p59OASL in various tissues
Tissue
Relative expression Number of blots where
tissue is represented
PBL ++++ 3
Stomach +++(+) 1
Spleen +++ 3
Lymph node +++ 1
Colon (muscosal lining) +++ 2
Fetal brain +++ 1
Thyroid ++(+) 1
Testis ++(+) 3
Thymus ++ 3
Fetal liver ++ 1
Pancreas ++ 1
Adrenal medulla ++ 1
Adrenal cortex ++ 1
Fetal kidney ++ 1
Uterus ++ 1
Small intestine + 3
Prostate + 2
Ovary + 1
Fetal lung
+ 1
A number of northerns were made using MTN filters from Clonetech (see text for
details). The relative expression was judged from the radiography after 5 days
exposure. RNA from some of the tissues was present on several filters, as indicated.
The functions of p59OASL
Escherichia coli expressed histidine-tagged p59OASL does not
have any 2–5A synthetase activity using conditions (44), where
an equivalent histidine-tagged p42OAS was fully active. This is
not surprising, taking into account the differences in the presumed
active sites, notably the P-loop.
There are evidently numerous experiments that have to be
performed to explore the functions of p59OASL, which could
by guest on June 1, 2013http://nar.oxfordjournals.org/Downloaded from
4127
Nucleic Acids Research, 1994, Vol. 22, No. 1
Nucleic Acids Research, 1998, Vol. 26, No. 18 4127
Figure 6. Regulation of the p59OASL gene by interferon. The induction of the
p59OASL gene by interferon-α or interferon-γ was measured by RNAse
protection assays, using total RNA form induced AMA cells. Cells were
induced as indicated. The protected fragments were separated on a 5% native
polyacrylamide gel, and the interesting bands were quantified using a
PhosphorImager. The p59OASL signal was normalised to the γ-actin signal
prior to calculation of the induction. Each column represents a mean of four
independent samples, the standard deviation is indicated.
constitute an important new regulatory feature in the 2–5A
pathway and connect this to the ubiquitin systems including the
proteasome control of cell cycle factors and transcription factors.
In particular, p59OASL could be a factor in the 2–5A involve-
ment in apoptosis (19,45).
ACKNOWLEDGEMENTS
We would like to thank the following people: Anette Bejder for
excellent help with the RACE experiment and Lars Aagaard for
giving invaluable help in using the Phylip package and for many
fruitful discussions, Niels Ole Kjeldgaard and Tracey Flint for
critical reading of the manuscript and many valuable comments,
Andrew King at the Sanger Centre (Cambridge, UK) and R. D.
Cox, Welcome Trust Centre for Human Genetics (Oxford, UK)
for valuable help with the genomic sequence and finally Cecilia
Rosada Kjeldsen for helpful discussion. This work was supported
by the Danish Natural Science Research Council (BIOTEK 3
program), The Danish Cancer Society and the Karen Elise
Jensen’s Foundation.
REFERENCES
1 Roberts,W.K., Hovanessian,A., Brown,R.E., Clemens,M.J. and Kerr,I.M.
(1976) Nature, 264, 477–480.
2 Hovanessian,A.G., Brown,R.E. and Kerr,I.M. (1977) Nature, 268, 537–540.
3 Clemens,M.J. and Williams,B.R. (1978) Cell, 13, 565–572.
4 Baglioni,C., Minks,M.A. and Maroney,P.A. (1978) Nature, 273, 684–687.
5 Williams,B.R., Saunders,M.E. and Willard,H.F. (1986)
Somatic Cell Mol. Genet., 12, 403–408.
6 Benech,P., Mory,Y., Revel,M. and Chebath,J. (1985) EMBO J., 4, 2249–2256.
7 Saunders,M.E., Gewert,D.R., Tugwell,M.E., McMahon,M. and
Williams,B.R. (1985) EMBO J., 4, 1761–1768.
8 Marie,I. and Hovanessian,A.G. (1992) J. Biol. Chem., 267, 9933–9939.
9 Chebath,J., Benech,P., Hovanessian,A., Galabru,J. and Revel,M. (1987)
J. Biol. Chem., 262, 3852–3857.
10 Dougherty,J.P., Samanta,H., Farrell,P.J. and Lengyel,P. (1980) J. Biol. Chem.,
255, 3813–3816.
11 Chebath,J. and Revel,M. (1992) In Baron,B., Copperhaver,F.,
Dianzani,W.R., Hughes,T.K.,Jr, Klimpel,G.R.,Jr, Niesel,D.W., Stanton,G.J.
and Tyring,S.K. (eds), Interferon: Principles and Medical Applications.
The University of Texas Medical Branch at Galverston, Galverston, TX,
pp. 225–236.
12 Hartmann,R., Noerby,P.L., Martensen,P.M., Joergensen,P., James,M.C.,
Jacobsen,C., Moestrup,S.K., Clemens,M.J. and Justesen,J. (1998)
J. Biol. Chem., 273, 3236–3246.
13 Johnston,M.I. and Torrence,P.F. (1984) In Friedman,R.M. (ed.),
Interferon 3. Mechanisms of Production and Action. Elsevier, Amsterdam,
pp. 189–298.
14 Chebath,J., Benech,P., Revel,M. and Vigneron,M. (1987) Nature, 330,
587–588.
15 Mitra,A., Higgins,D.W., Langenberg,W.G., Nie,H., SenGupta,D.N. and
Silverman,R.H. (1996) Proc. Natl Acad. Sci. USA, 93, 6780–6785.
16 Ogawa,T., Hori,T. and Ishida,I. (1996) Nature Biotechnol., 14, 1566–1569.
17 Rysiecki,G., Gewert,D.R. and Williams,B.R. (1989) J. Interferon. Res., 9,
649–657.
18 Narasimhan,J., Potter,J.L. and Haas,A.L. (1996) J. Biol. Chem., 271,
324–330.
19 Zhou,A., Paranjape,J., Brown,T.L., Nie,H., Naik,S., Dhong,B., Chang,A.,
Trapp,B., Fairchild,R., Colmenares,C. and Silverman,R.H. (1997) EMBO J.,
16, 6355–6363.
20 Silverman,R.H. (1997) Ribonucleases: Structure and Functions. Academic
Press, Inc., NY, pp. 515–551.
21 Marra,M.A., Hillier,L. and Waterston,R.H. (1998) Trends Genet., 14, 4–7.
22 Altschul,S.F., Gish,W., Miller,W., Myers,E.W. and Lipman,D.J. (1990)
J. Mol. Biol., 215, 403–410.
23 Frohman,M.A. (1994) PCR Meth. Appl., 4, S40–58.
24 Thompson,J.D., Higgins,D.G. and Gibson,T.J. (1994) Nucleic Acids Res.,
22, 4673–4680.
25 Felsenstein,J. (1996) Methods Enzymol., 266, 418–427.
26 Gunning,P., Ponte,P., Blau,H. and Kedes,L. (1983) Mol. Cell Biol., 3,
1985–1995.
27 Chomczynski,P. and Sacchi,N. (1987) Anal. Biochem., 162, 156–159.
28 Adams,M.D., Kerlavage,A.R., Fleischmann,R.D., Fuldner,R.A., Bult,C.J.,
Lee,N.H., Kirkness,E.F., Weinstock,K.G., Gocayne,J.D., White,O. et al.
(1995) Nature, 377, 3–174.
29 Kozak,M. (1995) Proc. Natl Acad. Sci. USA, 92, 2662–2666.
30 Kozak,M. (1997) EMBO J., 16, 2482–2492.
31 Lee,J.W., Choi,H.S., Gyuris,J., Brent,R. and Moore,D.D. (1995)
Mol. Endocrinol., 9, 243–254.
32 Yamagata,K., Oda,N., Kaisaki,P.J., Menzel,S., Furuta,H., Vaxillaire,M.,
Southam,L., Cox,R.D., Lathrop,G.M., Boriraj,V.V. et al. (1996) Nature,
384, 455–458.
33 Heinemeyer,T., Wingender,E., Reuter,I., Hermjakob,H., Kel,A.E., Kel,O.V.,
Ignatieva,E.V., Ananko,E.A., Podkolodnaya,O.A., Kolpakov,F.A.,
Podkolodny,N.L. and Kolchanov,N.A. (1998) Nucleic Acids Res., 26,
362–367.
34 Ghosh,S.K., Kusari,J., Bandyopadhyay,S.K., Samanta,H., Kumar,R. and
Sen,G.C. (1991) J. Biol. Chem., 266, 15293–15299.
35 Ghosh,A., Sarkar,S.N., Guo,W., Bandyopadhyay,S. and Sen,G.C. (1997)
J. Biol. Chem., 275, 33220–33226.
36 Ghosh,A., Desai,S.Y., Sarkar,S.N., Ramaraj,P., Ghosh,S.K., Bandyopadhyay,S.
and Sen,G.C. (1997) J. Biol. Chem., 272, 15452–15458.
37 Kon,N. and Suhadolnik,R.J. (1996) J. Biol. Chem., 271, 19983–19990.
38 Saraste,M., Sibbald,P.R. and Wittinghofer,A. (1990) Trends Biochem. Sci.,
15, 430–434.
39 Holm,L. and Sander,C. (1995) Trends Biochem. Sci., 20, 345–346.
40 Toniolo,D., Persico,M. and Alcalay,M. (1988) Proc. Natl Acad. Sci. USA,
85, 851–855.
41 Wiborg,O., Pedersen,M.S., Wind,A., Berglund,L.E., Marcker,K.A. and
Vuust,J. (1985) EMBO J., 4, 755–759.
42 Kas,K., Michiels,L. and Merregaert,J. (1992) Biochem. Biophys. Res.
Commun., 187, 927–933.
43 Banerji,J., Sands,J., Strominger,J.L. and Spies,T. (1990) Proc. Natl Acad.
Sci. USA, 87, 2374–2378.
44 Justesen,J., Ferbus,D. and Thang,M.N. (1980) Nucleic Acids Res., 8,
3073–3085.
45 Kisselev,L.L, Justesen,J., Wolfson,A.D. and Frolova,L.Y. (1998)
FEBS Lett., 427, 157–163.
by guest on June 1, 2013http://nar.oxfordjournals.org/Downloaded from
