domuncula col-lected from the sea display a considerable amount of 2–5A synthetase activity; 16% of the ATP substrate is converted to the 2–5A product, while tissue from specimens which
Trang 1Induction of (2¢ )5¢)oligoadenylate synthetase in the marine
by the bacterial endotoxin lipopolysaccharide
Vladislav A Grebenjuk1, Anne Kuusksalu2, Merike Kelve2, Joachim Schu¨tze1, Heinz C Schro¨der1
and Werner E G Mu¨ller1
1
Institut fu¨r Physiologische Chemie, Abteilung fu¨r Angewandte Molekularbiologie, Johannes Gutenberg-Universita¨t, Mainz, Germany;
2
Institute of Chemical Physics and Biophysics, Tallinn, Estonia
Recent studies have shown that the Porifera, with the
examples of the demosponges Suberites domuncula and
Geodia cydonium, comprise a series of pathways found also
in the immune system of Deuterostomia, such as vertebrates,
but are absent in Protostomia, with insects or nematodes as
examples One pathway is the (2¢)5¢)oligoadenylate
syn-thetase [(2–5)A synsyn-thetase] system In the present study we
show that crude extracts from tissue of S domuncula
col-lected from the sea display a considerable amount of (2–5)A
synthetase activity; 16% of the ATP substrate is converted to
the (2–5)A product, while tissue from specimens which were
kept for 6 months in an aquarium shows only 1% of
con-version As aquarium animals show a lower bacterial load,
those specimens were treated for the experiments with the
bacterial endotoxin lipopolysaccharide (LPS); they
respon-ded to LPS with a stimulation of the (2–5)A synthetase activity To monitor if this effect can be obtained also on the
in vitrolevel, primmorphs which comprise proliferating and differentiating cells, were incubated with LPS Extracts obtained from LPS-treated primmorphs also convert ATP
to the (2–5)A products mediated by the synthetase In par-allel to this effect on protein level, LPS causes after an incubation period of 12 h also an increase in the steady-state level of the transcripts encoding the putative (2–5)A syn-thetase It is postulated that in sponges the (2–5)A synthetase
is involved in antimicrobial defense of the animals Keywords: Suberites domuncula; Geodia cydonium (2¢-5¢) oligoadenylate synthetase; sponges; Porifera
Sponges (phylum Porifera) are with the other metazoan
phyla of monophyletic origin [1] These aquatic sessile filter
feeders existed already prior to the ÔCambrian explosionÕ [2],
which has been dated back 550 million years [3] This
implies that they must have developed powerful
mech-anisms to protect themselves against unfavorable conditions,
e.g environmental stress (ultraviolet exposure or
xenobiot-ics) [4,5] Because sponges have the capacity to process their
own volume of water every 5 s in order to extract edible
material [6] they are exposed to a huge amount of bacteria
and also viruses that are present in the seawater [7,8] To cope
with these threats, sponges have developed an efficient
chemical defense system [9] as well as humoral and cellular
defense mechanisms [10], that provided also the basis for the evolution to metazoan organisms [10]
One efficient protection against invading microorganisms
is the (2¢)5¢)oligoadenylate synthetase [(2–5)A synthetase] system [11–13] The (2–5)A synthetase(s) is activated by certain classes of RNA, mainly double-stranded RNA [14]
In vertebrates the (2–5)A pathway is also induced by interferons [15] The major enzyme in this pathway, the (2–5)A synthetase catalyzes the synthesis of a series of 2¢ )5¢-linked oligoadenylates, termed (2–5)A [¼ pppA(2¢p5¢A)n [pnAn], with chain lengths of 1£ n £ 30] from ATP [16,17] (2–5)A acts as an allosteric activator of a latent endoribonuclease, the RNase L, which degrades single-stranded, viral or cellular RNA [18]
Only very rarely viruses have been observed in sponges [19], while intracellular bacteria are frequently present [20] Some of the bacteria (both Gram positive and negative) found in sponges might act as symbionts [21], while others are presumably infectious [22] In a previous contribution
we demonstrated that sponges react to bacterial infection with suppression of cell proliferation and apoptosis [23]
In sponges the apoptotic pathway is well established
on molecular level; genes coding for both pro- (death domains-containing proteins) and anti-apoptotic proteins (Bcl-2 polypeptides) have been isolated from sponges [24,25] The (2–5)A synthetase-mediated inhibition of cell growth [26] as well as induction of apoptosis [27,28] have also been reported for vertebrate cells In addition it was demonstrated recently that besides the oligoadenylate synthesizing activity, the murine (2–5)A synthetase isoezyme
Correspondence to W E G Mu¨ller, Institut fu¨r Physiologische
Chemie, Abteilung Angewandte Molekularbiologie, Johannes
Gutenberg-Universita¨t, Duesbergweg 6, 55099 Mainz, Germany.
Fax: + 61 31 3925243, Tel.: + 61 31 3925910,
E-mail: wmueller@mail.uni-mainz.de
Abbreviations: LPS, lipopolysaccharide; CDP, disodium
2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2¢-(5¢-chloro)-tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate.
Note: The two cDNA sequences from Suberites domuncula for the
(2–5)A synthetase form 1, termed SD25A-1, and for the (2–5)A
synthetase form 2, termed SD25A-2, have been deposited in the
EMBL/GenBank database under accession numbers AJ301652 and
AJ301653, respectively.
(Received 26 September 2001, revised 7 January 2002, accepted
11 January 2002)
Trang 29-2 functions as a proapoptotic protein of the Bcl-2 family
[29]
Considering the fact that in sponges the molecules
involved in immune response are closer related to
deutero-stomian (vertebrate) animals than to Protostomia (insects or
nematodes; reviewed in [10]), we postulated that also
elements of the (2–5)A system exist in sponges The first
sponge species studied was Geodia cydonium
(Demospong-iae) which in fact showed high levels of (2–5)A
oligoade-nylate synthesis in comparison to vertebrate cells [30] The
reaction products were identified by thin-layer
chromatog-raphy, immunologically and by high-performance liquid
chromatography The biological activity of (2–5)A
oligo-mers was verified by inhibition of the protein synthesis in
rabbit reticulocyte lysate [30] The (2–5)A synthetase
reaction products were also confirmed by MALDI-MS
and by NMR analysis [31] We succeeded in cloning the
sponge (2–5)A synthetase from G cydonium [32] A
calcu-lation based on the rates of amino-acid substitutions
revealed that the sponge enzyme branched off from a
common ancestor 520 million years ago
In view of the finding that sponges do contain the (2–5)A
synthetase system like vertebrates, while this enzyme is
lacking in Protostomia [32] it was pressing to study in
functional assays if also in sponges the (2–5)A synthetase
responds in the protection against foreign, pathogenic
microorganisms The sponge cellular system, which proved
to be suitable for this approach are the sponge primmorphs
[33,34] Primmorphs are formed from dissociated single cells
after usually 5 days and reach sizes of 5 mm; they contain
proliferating cells and their interior is surrounded by an
almost complete single-cellular layer of epithelial-like cells,
pinacocytes; the cells inside the primmorphs are primarily
spherulous cells, amoebocytes and archaeocytes
In the present study, we use tissue and primmorphs from
the marine sponge Suberites domuncula (Demospongiae)
and tissue of G cydonium (Demospongiae; as a reference
sponge) and treated them with lipopolysaccharide (LPS), an
endotoxin derived from the outer cell wall of Gram-negative
bacteria that binds to the cell surface molecule CD14 [35]
The data revealed that tissue as well as primmorphs reacted
to LPS treatment with a rapid increase in (2–5)A synthetase
activity To determine if LPS has the same effect on the gene
expression level, two cDNAs that might encode the putative
(2–5)A synthetase have been isolated and characterized
from S domuncula Northern blot studies revealed that the
steady-state level of transcripts for the (2–5)A synthetase
gene strongly increased in tissue as well as in primmorphs
after LPS treatment Because LPS is known to strongly
inhibit protein synthesis in sponge cells [22], it is concluded
that the (2–5)A synthetase system is involved in defense
against microorganisms, very likely by inhibition of cell
proliferation or induction of apoptosis
M A T E R I A L S A N D M E T H O D S
Materials
Restriction endonucleases and other enzymes for
recombin-ant DNA techniques and vectors were obtained from
Stratagene (La Jolla, CA, USA), Qiagen (Hilden, Germany),
Roche (Mannheim, Germany), USB (Cleveland, OH, USA),
Amersham (Buckinghamshire, UK) and Promega
(Madi-son, WI, USA) In addition, DIG (digoxigenin) DNA labeling kit, DIG-11-dUTP, anti-DIG AP Fab fragments, disodium 2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2¢-(5¢-chloro)-tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate (CDP) and positively charged nylon membrane (no 1209272) were from Roche (Mannheim, Germany) and Na-hexafluorosilicate from Aldrich (Deisenhofen, Germany) Shrimp alkaline phosphatase was purchased from USB Corporation (Cleveland, OH, USA), [14C]ATP (542 mCiÆmmol)1) from Amersham International PLC (Buckinghamshire, England), LPS from Escherichia coli (L2880) and adenosine 5¢-triphosphate from Sigma Chemical
Co (St Louis, MO, USA), and polyethyleneimine cellulose TLC plates from Schleicher & Schuell (Keene, NH, USA) Rotiquant reagent was purchased from Roth (Germany) Sponge
Live specimens of S domuncula [Porifera, Demospongiae, Hadromerida], and G cydonium (Porifera, Demospongiae, Geodiidae) were collected by scuba diving near Rovinj (Croatia) from depths between 15 and 35 m (nonpolluted site) The sponges were brought to Mainz (Germany) and there kept in 1000-L tanks at 17°C before use in the experiments In one series of experiments the animals were kept for only 2 days in the aquarium before use (termed Ôsea animalsÕ) In the studies to determine the effect of LPS on the expression/activity of (2–5)A synthetase sponges remained for 6 months in the aquarium prior to use in the experiments (Ôaquarium animalsÕ; Fig 1A)
Formation of primmorphs The procedure for the formation of primmorphs from single cells was applied as described previously [33,34] Starting from single cells obtained by dissociation in Ca2+and Mg2+ -free artificial seawater [36] primmorphs of at least 1 mm in diameter (average, 3–7 mm) are formed after 2 days For the experiments, 5-day-old primmorphs were used They were cultivated in natural seawater supplemented with 0.2% of RPMI1640 medium and with silicate to the optimal concen-tration of 60 lM as described previously [37] The silicate concentration in the natural, nonsupplemented seawater was
1 lM The incubation temperature was set to 17°C The experiments were performed with six animals per assay, each Incubations
Sponges were cut into cubes with a side of approximately 0.5 cm For each set of experiments (exposure to LPS and control) samples from the same sponge specimen were used Incubations were performed in filtered, oxygenated sea-water The sponge cubes or primmorphs were treated for 0–
24 h in the absence or presence of 1 lgÆmL)1or 10 lgÆmL)1
of LPS in seawater at ambient temperature In control experiments the samples remained untreated for the entire incubation period Thereafter the sponge cubes/ primmorphs were immediately frozen ()80 °C)
Cell extracts Frozen sponge cubes were ground in liquid nitrogen and an equal amount (v/w) of the polymerase assay buffer (PAB)
Trang 3[20 mMTris/HCl, pH 7.5, containing 100 mMKCl, 5 mM
MgCl2and 5% (v/v) glycerol] was added during
homoge-nization The primmorphs were suspended in polymerase
assay buffer followed by 2 cycles of freezing ()10
°C)-thawing for lysis The supernatant obtained after
centrifu-gation (10 000 g; 10 min; 4°C) was immediately frozen
(Ôcrude extractÕ)
(2–5)A synthetase assay
(2–5)A synthetase activity in crude extracts was determined
after binding of the enzyme to a positively charged nylon
membrane The assays of the same series were normalized to
the protein content All incubations were performed in
microtiter plate wells at room temperature Sponge extract
was added to a piece of membrane (0.16 cm2) in the well
After incubation for 30 min with gentle shaking the
membrane was washed 4· 5 min with PAB and
subse-quently dried 10 lL of reaction buffer containing 1 mM
ATP and 2· 104c.p.m [14C]ATP, 30 mM Tris/HCl
pH 7.5, 100 mM KCl and 5 mM MgCl2 was added [38]
The wells were sealed tightly and the synthesis of (2–5)A was
allowed to occur usually for 4–12 h For HPLC analysis
50 lL of the reaction buffer without radioactive tracer was
used to produce the oligomers and the reactions were
performed in microcentrifuge tubes
Thin-layer chromatography
The reaction products were eluted with distilled water
and separated by TLC on polyethyleneimine cellulose using
0.4MTris/HCl pH 8.6, 30 mMMgCl2as the mobile phase
[30] The TLC plates were exposed to a CS-imaging screen
and scanned with the GS-525 Molecular Imager (Bio-Rad;
Hercules, CA, USA) The amounts of ATP and (2–5)A
oligomers were quantified by the relative intensities of the
corresponding spot areas on the autoradiograms
High-performance liquid chromatography
The 2¢-5¢ linked oligoadenylates produced in the assay in
their triphosphorylated forms were applied to the HPLC
column (Supelcosil LC-18, 30 cm· 4 mm, 5 lm; Supelco) and separated in a 0.5–30% methanol gradient in 50 mM
NH4H2PO4 pH 7.0 at 40°C [39] The absorbance was measured at 254 nm The 3.05 software version (Waters Corporation) was used to quantify the synthesis products
Dephosphorylation of (2–5)A oligomers The synthesis products of S domuncula were verified and quantified as the dephosphorylated (ÔcoreÕ) forms of the oligomers For that purpose the mixture of products was treated with shrimp alkaline phosphatase 0.04 UÆlL)1for
1 h at 37°C followed by the inactivation of the enzyme for 15 min at 65°C After centrifugation at 20 000 g for
10 min, the mixture was applied to the HPLC column
Cloning of the putative S domuncula (2–5)A synthetase cDNA
Two complete sponge cDNAs, termed SD25A, encoding the putative (2–5)A synthetase (25A_SD), were cloned by screening the cDNA library from S domuncula [40] using the GC2–5AS (accession number Y18497 [32]) as a probe Screening of the library was performed under low stringency hybridization as described previously [40] Positive clones were detected with an alkaline phosphatase conjugated anti-DIG Ig using 5-bromo-4-chloroindol-2-yl phosphate/nitro blue tetrazolium as substrate [41] All cDNAs have been obtained from two different cDNA libraries resulting in three independent clones each DNA sequencing was performed with an automatic DNA sequenator (Li-Cor 4000S) Two different complete sequences have been obtained; they were termed SD25A-1 and SD25A-2 The corresponding deduced proteins were named 25A-1_SD and 25A-2_SD Sequence comparisons
The sequences were analyzed using computer programs BLAST [42] and FASTA [43] Multiple alignments were performed withCLUSTAL Wver 1.6 [44] Phylogenetic trees were constructed on the basis of amino-acid sequence
Fig 1 S domuncula: animals and prim-morphs (A) The siliceous sponge S domun-cula (red) has been kept for more than
6 months together with the second demo-sponge D avara (violet) in the aquarium (·0.1) (B–D) Primmorph formation of
S domuncula (B) Dissociated single cells (·200) (C) Primmorphs formed after 5 days; (·5) (D) Cross section through a primmorph, which has been subsequently subjected to incubation with antiserum raised against
S domuncula cells (·5).
Trang 4alignments by neighbour-joining, as implemented in the
NEIGHBOR program from the PHYLIP package [45] The
distance matrices were calculated using the Dayhoff PAM
matrix model as described previously [46] The degree of
support for internal branches was further assessed by
bootstrapping [45] The graphic presentations were
pre-pared withGENEDOC[47] Hydropathicity analysis, based on
the method of Kyte & Doolittle [48], was performed using
thePC/GENESoap [49]
Exposure of primmorphs toEscherichia coli
Primmorphs (5 days old), obtained from cells of aquarium
animals, were exposed to heat-killed Escherichia coli as
described earlier [23]; the concentration of bacteria was
adjusted to 10 lg of nitrogen per mL [23] Twelve and
twenty-four hours later primmorphs were taken and RNA
was extracted which then was subjected to Northern
blotting, using SD25A-1 as a probe
Northern blot
RNA was extracted from liquid-nitrogen pulverized sponge
tissue with TRIzol Reagent (GibcoBRL, Grand Island,
NY, USA) Then an amount of 5 lg of total RNA was
electrophoresed through 1% formaldehyde/agarose gel and
blotted onto Hybond N+membrane following the
manu-facturer’s instructions (Amersham; Little Chalfont,
Buck-inghamshire, UK) [5] Hybridization was performed with a
0.7-kb part of SD25A-1 The probe was labeled with the
PCR-DIG-Probe-Synthesis Kit according to the
manufac-turer’s instructions (Roche) In one series of experiments
poly(A)+-RNA was purified from sponge tissue with
Oligotex mRNA kit (Qiagen) and analysed For the
quantification of the Northern blot signals the
chemilumi-nescence procedure was applied [50]; CDP-Star was used as
substrate The screen was scanned with the GS-525
Molecular Imager (Bio-Rad)
Immunohistological analysis of primmorphs
Fresh tissue was fixed in paraformaldehyde, embedded in
Technovit 8100 and sectioned, essentially as described
previously [23] The 2-lm thick slices reacted with
anti-serum, raised against S domuncula cells
The polyclonal antiserum against cells from S domuncula
was raised in female rabbits (White New Zealand) An
amount of 3· 106cells [34] was injected at 4-week intervals;
after three boosts, serum was prepared [51] The antiserum
obtained was termed anti-S domuncula
After fixation of the slices from S domuncula
prim-morphs, the cells were made permeable with 0.1% saponin,
washed in NaCl/Pi and incubated with anti-S domuncula
for 30 min and finally with fluorescein
isothiocyanate-conjugated goat anti-(rabbit IgG) Ig for 2 h [52] The sections
were inspected by immunofluorescence with an Olympus
AHBT3 microscope Control experiments with preimmune
serum did not show any auto- immunofluorescence
Protein quantification
Protein concentration was estimated by the Rotiquant
reagent, using bovine serum albumin as a standard
R E S U L T S
Identification of (2–5)A synthetase activity
inS domuncula ‘crude extract’
It is established that (2–5)A synthetase is present in
G cydoniumin high amounts The product length formed
by the G cydonium enzyme from ATP is 2–8 adenylate residues and the 2¢)5¢ linkage was verified by NMR analysis [31] The p3A4proved to be biologically active [30] It also appears that G cydonium is not the only species with (2–5)A synthesizing activity within the phylum Porifera We have identified (2–5)A synthesizing activity in different marine sponges, including S domuncula (this study and A Kuusksalu, A Lopp, T Reintamm and H Kelve, unpub-lished data) The product pattern of the enzyme from
S domuncula differs under the same reaction conditions from that of G cydonium The synthesis level is significantly lower and the main synthesis product is p3A2as verified by the comigration with p3A2standard (TLC and HPLC) and
A2standard (HPLC) after dephosphorylation with shrimp alkaline phosphatase (not shown) Recently we have shown
in G cydonium as well as in S domuncula crude extracts that the enzyme, catalyzing the formation of (2–5)A, does not require dsRNA for activity (submitted) In the present study we took advantage of this phenomenon and per-formed the assays for (2–5)A synthesis with crude cell extracts using positively charged membranes for partial purification of the enzymes
(2–5)A synthetase activity from field/aquarium animals Interestingly, samples from S domuncula kept in the aquarium for 6 months had lower (2–5)A synthesizing activities compared to those which were cut into pieces and frozen after only 2 days maintenance in an aquarium The extract from aquarium animals converted, under otherwise identical conditions, only 1% of the substrate to the (2–5)A product; in contrast extracts from the sea animals could utilize more than 16% of the substrate during the same synthesis period (Fig 2; Table 1)
In the case of G cydonium the synthetase activity was initially high in all animals tested (total product formation
at the same reaction conditions was 82.8% in sea animals and 33.6% in aquarium animals), still revealing significant product decrease during the long-term incubation (for
6 months) in the aquarium (Table 2)
This result suggested that the animals kept in the aquarium are lacking a factor that causes either the expression of the gene encoding the (2–5)A synthetase or the activation of the enzyme One potential factor could be the differential load of microbes In a recent study it could
be established that specimens from S domuncula, analyzed immediately after being taken from the sea, harbor a series
of bacterial strains (> five strains; very likely commensalic ones), while those which were kept for 6 months in an aquarium contained only one bacterial strain (likely to be the symbiotic one) which showed high rRNA sequence similarity to a Pseudomonas species The latter bacterial species was operationally termed S domuncula symbiont (GenBank accession number AF324886 [22]) These sym-bionts were found to be encapsulated inside special cells, the bacteriocytes, present in the vicinity of the canals This result
Trang 5was taken as the rationale to study if lipopolysaccharide
(LPS), a known endotoxin derived from the outer cell wall
of gram-negative bacteria, may influence the activity of the
(2–5)A synthetase
Effect of LPS on (2–5)A synthetase activity in tissues
fromS domuncula and G cydonium
Tissue samples from S domuncula specimens, kept for
6 months in the aquarium are almost devoid of (2–5)A
synthetase activity, under the conditions used
Approxi-mately 1% of the substrate was converted to (2–5)A
oligomers during 12 h synthesis Tissue from these animals
was used to analyze if the endotoxin LPS has the capacity to
induce the enzyme The data revealed that in the presence of
1 lgÆmL)1of LPS the (2–5)A synthetase activity started to
increase; after 3–12 h incubation period 4% of the ATP
substrate was converted to p3A2 (Table 1) This increase
was transient and during longer incubation periods (24 h)
the product level dropped again Higher concentrations of
LPS (10 lgÆmL)1) caused a lower effect on the (2–5)A
synthesizing activity in S domuncula
Despite the initially high (2–5)A synthesizing activity in tissue of G cydonium (aquarium animals), the incubation with LPS caused significant increase of synthesis level (Table 2) The time course of the induction showed similarity to the effect we had seen in the case of
S domuncula The increment of the products was highest
on the third hour of incubation (1 lgÆmL)1LPS) The most drastic increase (3 h; 1 lgÆmL)1LPS) could lead almost to the synthesis level of the sea animal Longer incubation caused again a decrease of the synthesis level Incubation at
10 lgÆmL)1of LPS gave a lower effect on synthetase activity than at 1 lgÆmL)1
Effect of LPS on (2–5)A synthetase activity
in primmorphs fromS domuncula Primmorphs were prepared from single cells (Fig 1B) of aquarium animals and used 5 days later for the experiment (Fig 1C) In order to make certain that the cells which had been reorganized into primmorphs indeed originated from the S domuncula species, cross sections through the cells were reacted with anti-(S domuncula) serum The immu-nofluorescence analysis shows that all (> 95% of the total) cells included in the primmorphs were stained brightly with the antiserum (Fig 1D) Control sections, incubated with preimmune serum did not show any reaction
The experiments show again that after incubation of the primmorphs with 1 lgÆmL)1of LPS for 3 h an increase of (2–5)A synthetase activity can be measured (from 1.5% (controls) to 3.3% of the ATP substrate was converted to
p3An after this period), Table 1 This amount does not change significantly during a prolonged incubation for up to
24 h The identity of the p3A2product synthesized by the (2–5)A synthetase both in tissue and in primmorphs of
S domunculawas verified by TLC and HPLC analysis as triphosphorylated and/or core oligomers
Two CDNAs encoding the putativeS domuncula (2–5)A synthetase
Two cDNAs, named SD25A-1 (accession number AJ301652) and SD25A-2 (AJ301653), have been isolated which comprise 1175 and 1205 nucleotides The longest ORFs translate to 324 amino acids (for the predicted polypeptide 25A-1_SD) and to 322 amino acids (25A-2_SD), respectively; Fig 3A The start ATG for 25A-1_SD
is located at nucleotides 62–64 (stop codon, nucleotides 1034–1036) and for 25A-2_SD at nucleotides 62–64 (nucle-otides 1028–1030) Northern blot analyses showed that the transcript length for SD25A-1 is 1.4 kb (see below) and for SD25A-21.3 kb (not shown), indicating that the full length clones have been isolated The calculated relative molecular masses for these new synthetases are 37 846 and 37 494, respectively The two proteins were predicted to be unstable with instability indices of 40.1 1_SD) and 46.0 (25A-2_SD), respectively [49]
The two sponge sequences show the characteristic domains, found in other (2–5)A synthetases, from the sponge G cydonium and in mammals (mouse) and chicken: The (2–5)A synthetase signature-1 [14], is found between amino acid 195 and amino acid 206 and signature-2 between amino acid 258 and amino acid 268; the positions refer to
Fig 2 Autoradiogram of the thin layer chromatography of the [14C]
labelled 2¢-5¢ oligoadenylates synthesized as described under Materials
and methods Lane a: S domuncula (aquarium animal); lane b:
S domuncula (sea animal); lane c: G cydonium (sea animal) Lane a
and b: 6 mg of total protein per assay, synthesis time 12 h; lane c: 2 mg
of protein, synthesis time 3.5 h Reaction products were separated by
polyethyleneimine cellulose TLC followed by visualization with
GS-525 Molecular Imager System The position of the authentic
compounds (AMP, ADP, ATP as well as p 3 A 2 and p 3 A 3 ) which were
run in parallel is shown.
Trang 6the 25A-1_SD sequence (Fig 3A) The ATP-binding site
essential for enzyme activity [53,54] resides between amino
acid 273 and amino acid 284 The dsRNA binding region of
(2–5)A synthetase has been narrowed down to the segment
within amino acid 104 and amino acid 158 of the murine
enzyme [53]; in S domuncula a related stretch has been
found between amino acid 76 and amino acid 125 The
polyA-related domain, found in enzymes such as poly(A)
polymerase (2–5)A synthetase and topoisomerase 1
(acces-sion number IPR001201 [55]), spans from amino acid 148
and amino acid 212
Phylogenetic analysis of sponge (2–5)A synthetases
Based on sequence similarity no sequence related to (2–5)A
synthetases from sponges or from vertebrates, is present in
the Protostomia Caenorhabditis elegans or Drosophila melanogaster (Advanced BLAST available from http:// www.ncbi.nlm.nih.gov/blast/blast.cgi); a related enzyme is also lacking in yeasts (e.g Saccharomyces cerevisiae) or plants (Arabidopsis thaliana) The two S domuncula sequences share with each other 95% identity and 97% similarity with respect to amino acids and with the
G cydonium enzyme 28% identity and 48% similarity The percent identity (similarity) to the mammalian (mouse) (2–5)A synthetase is 19% (36%) and to the chicken sequence 19% (35%); Fig 3A
A phylogenetic tree was constructed on the basis of amino-acid sequence alignments (Fig 3B) by neighbour-joining of the vertebrate and sponge (2–5)A synthetases The distantly related sequence of anthocyanidin synthase from the plant Dianthus caryophyllus (U82432) was used as
Table 1 Determination of (2–5)A synthetase activity in tissue (both from sea animals and aquarium animals) and primmorphs from S domuncula (obtained from aquarium animals) The samples were incubated with 1 or 10 lgÆmL)1of LPS for a period of 0–24 h Subsequently crude extracts were prepared and reacted in the enzyme assay with ATP (12 h synthesis time for the tissue extracts; 22 h for primmorph extracts) as described under Materials and methods The products were dephosphorylated after synthesis with shrimp alkaline phosphatase and analysed by HPLC The reaction products were also analyzed by TLC, followed by autoradiography to determine the product Based on these data the conversion of [ 14 C]ATP to (2–5)A was calculated and is given in percent to the sum of ATP, ADP and AMP (The SD is less than 15%; n ¼ 5).
Animals
LPS (lgÆmL)1)
Incubation period (h)
ATP + ADP + AMP (%)
Product (%)
S domuncula tissue
S domuncula primmorphs
Table 2 Determination of (2–5)A synthetase activity in tissue (from sea animals and aquarium animals) of G cydonium Where indicated, incubation with 1 or 10 lgÆmL)1of LPS was performed for 0–24 h Extracts were prepared, reacted in the enzyme assay with ATP for 3.5 h, the products were analysed by HPLC as described in Materials and methods The amount of 2¢)5¢ linked dimers (p 3 A 2 ) as well as trimers (p 3 A 3 ) and longer were calculated based on corresponding peak areas (The SD is less than 15%; n ¼ 5).
Animals
LPS (lgÆmL)1)
Incubation period (h)
ATP + ADP + AMP (%)
p 3 A 2
(%)
p 3 A 3 and longer (%)
Product (sum %)
Trang 7outgroup to root the tree The anthocyanidin synthase is
known to be involved in the catalysis of the colorless
leucoanthocyanidins to the colored anthocyanidins [56]
The phylogenetic relationship reveals that the three sponge sequences form the basis of the tree from which the vertebrate sequences branch off
Fig 3 The two putative sponge (2–5)A synthetases from S domuncula (A) Alignment of the amino-acid sequence of the two sponge sequences, 25A-1_SD and 25A-2_SD, deduced from the cDNAs SD25A-1 and SD25A-2, with the related proteins from the sponge G cydonium (25A_GEOCY, accession number Y18497), as well as from mouse (25A_MOUSE, P11928) and from chicken (25A_CHICK, AB002586) The alignment was performed using the CLUSTAL W program Residues of amino acids, similar among all sequences, are in inverted type and residues conserved in at least three sequences are shaded The characteristic signatures of the (2–5)A synthetase are indicated: the two conserved signatures (| Sig-1 and | Sig-2), the potential ATP-binding region (|+ ATP), the dsRNA binding segment (|– Bdg: dsRNA) and the polyA-related domain (|::: polyA-related domain) (B) The phylogenetic relationship of the five (2–5)A synthetase sequences The tree was routed with the distantly related sequence of anthocyanidin synthase from the plant Dianthus caryophyllus (ANTO_DC, U82432) The numbers at the nodes are an indication of the level of confidence for the branches as determined by bootstrap analysis (1000 bootstrap replicates) The scale bar indicates an evolutionary distance
of 0.1 amino-acid substitutions per position in the sequence.
Trang 8Increase in the steady-state level of the (2–5)A
synthetase transcripts by LPS
The effect of LPS on the steady-state level of (2–5)A
synthetase transcripts, SD25A-1, was monitored by
Nor-thern blotting in a semiquantitative way both in tissue from
aquarium animals as well as in primmorphs obtained from
them The data show that in tissue from those animals no
expression could be visualized after blotting with the
SD25A-1 probe (Fig 4A, lane a) However, after an
incubation period for 12 h in the presence of 1 lgÆmL)1of
LPS, a clear 1.4-kb band became visible which reflects the
size of the (2–5)A synthetase gene (Fig 4A, lane b) A
likewise strong expression is also seen if the poly(A)+-RNA
fraction from tissue, exposed to LPS for the same period,
was subjected for Northern blotting (Fig 4B, lane a); in
contrast poly(A)+-RNA from nontreated tissue did not
show any signal (not shown)
If single cells, kept for 3 days in Ca2+- and Mg2+-free
artificial seawater (Fig 4B, lane b), or primmorphs, not
treated with LPS (Fig 4B, lane c) were analyzed for
transcripts of (2–5)A synthetase, no signal in the 1.4-kb size
range could be seen However, if the primmorphs were
treated with 1 lgÆmL)1 LPS a strong expression of the
(2–5)A synthetase gene is seen after 12 h (Fig 4B, lane d);
the 1.4-kb signal even increased if RNA was analyzed from
primmorphs, incubated with LPS for 24 h (Fig 4B, lane e)
Increase in the steady-state level of the (2–5)A
synthetase transcripts during incubation withE coli
In view of our earlier finding that S domuncula cells
respond to exposure to heat-killed E coli with a reduced cell
proliferation and cell viability [23], primmorphs were
exposed to dead bacteria under the conditions described
The bacteria were added at a concentration of 10 lg of
nitrogen per mL to the primmorphs; 12 and 24 h later RNA
was extracted and then probed with the SD25A-1 cDNA in
the Northern blotting experiment
The results show that in the absence of the heat-killed
bacteria no transcripts, corresponding to 1.4 kb (2–5)A
synthetase mRNA, can be identified in the Northern blotting
approach (Fig 4C, lanes a to c) In contrast, the steady-state
level of the transcripts increased strongly, even after the short
incubation period of 12 h (Fig 4D, lane b vs lane a; at time
0) A prolonged incubation for 24 h resulted in an even higher
level of the (2–5)A synthetase transcripts (Fig 4C, lane c)
D I S C U S S I O N
Inhibition of cell growth, apoptosis and inhibition of
protein synthesis are ways of protection of metazoan
organisms against death caused by microbes The bacterial
endotoxin LPS causes cell growth inhibition [57] as well as
induction of apoptosis [27] in vertebrates very likely via a
(2–5)A synthetase-mediated pathway Also in sponges LPS
inhibits cell proliferation and apoptosis [22,23]
Further-more, LPS strongly inhibits protein synthesis in S
domun-cula[22] Therefore, in the present study we tried to answer
the question of whether also this effect is mediated or
paralleled by a stimulation/induction of (2–5)A synthetase
in sponges, using two different sponge species, S domuncula
and G cydonium as examples
Tissue samples from S domuncula and G cydonium displayed different (2–5)A synthetase activities depending
on the time of cultivation in the aquarium If crude extracts from animals were taken (almost) immediately out of the sea (sea animals) which were analyzed for (2–5)A synthetase activity, product was detectable In the present study this effect was documented for S domuncula and G cydonium (Tables 1 and 2) If these animals (S domuncula) were kept
Fig 4 Effect of LPS and heat-killed E coli on the steady-state level of (2–5)A synthetase transcripts in S domuncula tissue from aquarium animals (A) and primmorphs/cells (B) Sponge tissue or primmorphs were incubated with 1 lgÆmL)1of LPS for 0–12 h Thereafter RNA was isolated and Northern blotting was performed with SD25A-1 to determine the expression of the (2–5)A synthetase gene Five micro-grams of total RNA each were loaded on the slot In one series of experiments single cells which remained in Ca2+- and Mg2+-free artificial seawater for 3 days were used to isolate RNA which was subjected to Northern blotting (B; lane b) The incubation period for the tissue was 0 (A; lane a) and 12 h (A; lane b); the primmorphs were incubated for 0 h (B; lane c), 12 h (lane d) or 24 h (lane e) M, marker RNAs, which were run in parallel In (B; lane a) poly(A) + -RNA, isolated from tissue of an aquarium animals, which was treated with
1 lgÆmL)1of LPS for 12 h, was analysed Determination of the effect
of heat-killed E coli on the steady-state level of (2–5)A synthetase mRNA in primmorphs Primmorphs were incubated in the absence (C) or presence of of the heat-killed bacteria (D) for 0–24 h At the indicated times primmorphs were taken, RNA was extracted and subjected at the same concentrations (5 lg) to Northern blotting experiments using the SD25A-1 cDNA as a probe.
Trang 9for a longer period, more than 6 months, in the aquarium
(aquarium animals) almost no enzyme activity was observed
(Fig 2; Tables 1 and 2) One reason for this effect is the fact
that the bacterial load, with respect to the number as well as
the species diversity of bacteria, is reduced under the
controlled aquarium conditions (closed circuit) The
reduc-tion of the bacterial flora in specimens kept in the aquarium
has been recently documented [22]
To test the assumption that bacterial load of sponge
tissue is causatively connected with (2–5)A synthetase
activity, the endotoxin LPS from the outer bacterial cell
wall was used as a substitution/model component
Incuba-tion studies with tissue from aquarium animals (S
domun-cula, G cydonium) revealed that LPS causes a significant
and rapid stimulation of the synthetase activity The extent
of products formed in S domuncula amounts to 1–2% of
conversion of ATP to (2–5)A, in comparison to 16%
measured in field sea animals while the corresponding
values for G cydonium had the same tendency It should be
mentioned that the (2–5)A synthesizing activity in G
cydo-niumis per se markedly higher than that in S domuncula
This stimulatory effect of LPS on the (2–5)A synthetase
activity was confirmed using the primmorph system from
S domuncula The primmorphs that contain proliferating
and differentiating cells [33,34] have been demonstrated here
to consist almost exclusively of sponge, S domuncula, cells
These experiments were included in order to rule out the
possibility that nonsponge cells form the aggregates
Previ-ously it had been argued that contaminating unicellular
eukaryotic organisms could have formed the aggregates that
might have been erroneously contributed to sponge cells
[58] The antiserum raised against S domuncula cells was
found to stain the cells of the primmorphs brightly Using
this primmorph system, it was demonstrated that again after
the incubation with LPS a significant amount of (2–5)A is
synthesized; 3.5% of the ATP present in the assays was
converted to dimers which comigrate with p3A2if analyzed
by TLC or coelute with the reference compound in HPLC
runs
Based on the incubation studies with tissue samples or
primmorphs it could be deduced that LPS causes a
stimulation of (2–5)A synthetase activity by a hitherto
unknown signal transduction pathway In a previous study
it had been shown that the mitogen-activated protein kinase
pathway is involved in the cell response to LPS [22] Until
now a potential involvement of this pathway in the (2–5)A
synthetase system has not been reported Nonetheless,
the fast response of the cells to LPS argues in favor of a
post-translational/allosterical activation of the (2–5)A
synthetase
The effect of LPS on the steady-state level of the
S domuncula (2–5)A synthetase transcripts was analyzed
in tissue and primmorphs, incubated with LPS and
heat-killed bacteria The results revealed that the steady-state
level of the transcripts is strongly up-regulated after an at
least 12-h incubation period This finding supports the
view that LPS causes not only a post-translational/
allosteric activation of the (2–5)A synthetase activity in
cells and tissue but also an increased transcript level The
potency of LPS to modulate gene expression in vertebrate
cells is well established [58]; nevertheless, the involvement
of the toxin in the (2–5)A synthetase pathway in these
systems has not yet been described However, the
partici-pation of LPS in apoptosis has been documented as reviewed recently [59]
Even though the documentation of virus infection/ presence in sponges is very poor in contrast to that of bacterial association/infection, which is very abundant in Demospongiae, the data presented show that the activity of the enzyme as well as the steady-state level of the transcripts
of the respective gene increases in cells after LPS/bacteria treatment Therefore, we currently subscribe to the view that LPS affects two pathways, one which causes a post-translational/allosteric activation of the enzyme resulting
in the formation of the p3Anproducts and a second, that increases the steady-state level of the transcripts of the corresponding (2–5)A synthetase gene In vertebrate cells it has been demonstrated that the expression of the (2–5)A synthetase is mediated by the jak/STAT pathway and initiated by cytokines [28] At present, studies on the elucidation of this pathway in S domuncula are in progress
in our group As a consequence of the activation/induction
of (2–5)A synthetase the sponge specimens might protect themselves against microbial infection or inhibition of cell proliferation and finally may undergo apoptosis The existence of cytokines in sponges has been documented, e.g the macrophage-derived cytokine-like molecule (the allograft inflammatory factor or glutathione peroxidase) or the polypeptide related to the mammalian endothelial-monocyte-activating polypeptide (reviewed in [10])
In conclusion, the data reported here suggest that the products of the (2–5)A synthetase in sponges, p3An, could
be involved in the antimicrobial defense of the animals Furthermore, sequence data show that genes encoding a putative (2–5)A synthetase are present in different sponge species This adds further support for the view that the immune system in sponges is closer related to the deutero-stomian, vertebrate, taxa than to the protostomian systems [60], which are lacking not only a series of characteristic cytokines [61] but also the (2–5)A synthetase system Future transfection studies must show if the genes encoding the putative (2–5)A synthetases from S domuncula are indeed responsible for the (2–5)A synthetase activity measured in cells from S domuncula
A C K N O W L E D G E M E N T S
This work was supported by grants from the Deutsche Forschungs-gemeinschaft (Mu¨ 348/14-1), the European Commission (project: SPONGE), the Bundesministerium fu¨r Bildung und Forschung (project: Center of Competence BIOTEC-MARIN), the International Human Frontier Science Program (RG-333/96-M) and the Estonian Science Foundation.
R E F E R E N C E S
1 Mu¨ller, W.E.G (1995) Molecular phylogeny of Metazoa (ani-mals): monophyletic origin Naturwiss 82, 321–329.
2 Mehl, D., Mu¨ller, I & Mu¨ller, W.E.G (1998) Molecular biolo-gical and palaeontolobiolo-gical evidence that Eumetazoa, including Porifera (sponges), are of monophyletic origin In Sponge Science – Multidisciplinary Perspectives (Watanabe, Y & Fuse-tani, N., eds), pp 133–156 Springer-Verlag, Tokyo.
3 Bengtson, S (1998) Animal embryos in deep time Nature 391, 529–530.
4 Batel, R., Hassanein, H.M.A., Schro¨der, H.C & Mu¨ller, W.E.G (1998) Increased expression of the sponge, Geodia cydonium,
Trang 10homolog of the human XPB/ERCC-3 excission DNA repair gene
after exposure to DNA-damaging chemical agents Mutation Res.
409, 123–133.
5 Wiens, M., Koziol, C., Hassanein, H.M.A., Batel, R & Mu¨ller,
W.E.G (1998) Expression of the chaperones 14-3-3 and HSP70
induced by PCB 118 (2,3¢,4,4¢,5-pentachlorobiphenyl) in the
mar-ine sponge Geodia cydonium Mar Ecol Progr Ser 165, 247–257.
6 Vogel, S (1977) Current-induced flow through living sponges in
nature Proc Natl Acad Sci USA 74, 2069–2071.
7 Gonzales, J.M & Moran, M.A (1997) Numerical dominance
of a group of marine bacteria in the alpha-subclass of the class
Proteobacteria in coastal seawater Appl Environ Microbiol 63,
4237–4242.
8 Kennish, M.J (1994) Practical Handbook of Marine Science CRC
Press, Boca Raton, FL.
9 Proksch, P (1994) Defensive role for secondary metabolites from
marine sponges and sponge-feeding nudibranchs Toxicon 32,
639–655.
10 Mu¨ller, W.E.G., Blumbach, B & Mu¨ller, I.M (1999) Evolution of
the innate and adaptive immune systems: relationships between
potential immune molecules in the lowest metazoan phylum
[Porifera] and those in vertebrates Transplantation 68, 1215–1227.
11 Lengyel, P.A.R.B (1982) Biochemistry of interferons and their
action Annu Rev Biochem 51, 251–282.
12 Sen, G.C & Lengyel, P (1992) The interferon system: a bird’s eye
view of its biochemistry J Biol Chem 267, 5017–5022.
13 Rebouillat, D., Hovnanian, A., Marie, I & Hovanessian, A.G.
(1999) The 100-kDa 2¢,5¢-oligoadenylate A synthetase catalyzing
preferentially the synthesis of dimeric ppp2¢pA5¢ molecules is
composed of three homologous domains J Biol Chem 274,
1557–1565.
14 Hartmann, R., Noerby, P.L., Martensen, P.M., Joergensen, P.,
James, M.C., Jacobson, C., Moestrup, S.K., Clemens, M.J &
Justesen, J (1998) Activation of 2–5 oligoadenylate synthetase by
single-stranded and double-stranded RNA aptamers J Biol.
Chem 273, 3236–3246.
15 Pestka, S., Langer, J.A., Zoon, K.C & Samuel, C.E (1987)
Interferons and their actions Annu Rev Biochem 56, 727–777.
16 Chebath, J., Benech, P., Hovanessian, A.G., Galabru, J., Robert,
N & Revel, M (1987) Four different forms of interferon-induced
2¢,5¢-oligo(A) synthetase identified by immunoblotting in human
cells J Biol Chem 262, 3852–3857.
17 Hovanessian, A.G (1991) Interferon-induced and
double-stran-ded RNA-activated enzymes: a specific protein kinase and
2¢,5¢-oligoadenylate synthetases J Interferon Res 11, 199–205.
18 Zhou, A., Hassel, B.A & Silverman, R.H (1993) Expression
cloning of 2–5A-dependent RNAase: an uniquely regulated
mediator of interferon action Cell 72, 753–765.
19 Vacelet, J & Gallissian, M.F (1978) Virus-like particles in cells of
the sponge Verongia cavernicola (Demospongiae) Dictyoceratida
and accompanying tissue changes J Invert Pathol 31, 246–254.
20 Simpson, T.L (1984) The Cell Biology of Sponges
Springer-Verlag, New York.
21 Althoff, K., Schu¨tt, C., Steffen, R., Batel, R & Mu¨ller, W.E.G.
(1998) Evidence for a symbiosis between bacteria of the genus
Rhodobacter and the marine sponge Halichondria panicea: harbor
also for putatively-toxic bacteria? Mar Biol 130, 529–536.
22 Bo¨hm, M., Hentschel, U., Friedrich, A., Fieseler, L., Steffen, R.,
Gamulin, V., Mu¨ller, I.M & Mu¨ller, W.E.G (2001) Molecular
response of the sponge Suberites domuncula to bacterial infection.
Mar Biol 139, 1037–1045.
23 Wagner, C., Steffen, R., Koziol, C., Batel, R., Lacorn, M.,
Steinhart, H., Simat, T & Mu¨ller, W.E.G (1998) Apoptosis in
marine sponges: a biomarker for environmental stress (cadmium
and bacteria) Mar Biol 131, 411–421.
24 Wiens, M., Krasko, A., Mu¨ller, C.I & Mu¨ller, W.E.G (2000)
Molecular evolution of apoptotic pathways: cloning of key
domains from sponges (Bcl-2 homology domains and death domains) and their phylogenetic relationships J Mol Evol 20, 520–531.
25 Wiens, M., Krasko, A., Mu¨ller, I.M & Mu¨ller, W.E.G (2000) Increased expression of the potential proapoptotic molecule DD2 and increased synthesis of leukotriene B 4 during allograft rejection
in a marine sponge Cell Death Diff 7, 461–469.
26 Rysiecki, G., Gewert, D.R & Williams, B.R (1989) Constitutive expression of 2¢,5¢-oligoadenylate synthetase cDNA results in increased antiviral activity and growth suppression J Interferon Res 9, 649–657.
27 Zhou, A., Paranjape, J., Brown, T.L., Nie, H., Naik, S., Dhong, S., Chang, A., Trapp, B., Fairchild, R., Colmenares, C & Silverman, R.H (1997) Interferon action and apoptosis are defective in mice devoid of 2¢,5¢-oligoadenylate-dependent RNase
L EMBO J 16, 6355–6363.
28 Justesen, J., Hartmann, R & Kjeldgaard, N.O (2000) Gene structure and function of the 2¢-5¢-oligoadenylate synthetase family Cell Mol Life Sci 57, 1593–1612.
29 Ghosh, A., Sarkar, S.N., Rowe, T.M & Sen, G.C (2001)
A specific isozyme of 2¢-5¢ oligoadenylate synthetase is a dual function proapoptotic protein of the Bcl-2 family J Biol Chem.
276, 25447–25455.
30 Kuusksalu, A., Pihlak, A., Mu¨ller, W.E.G & Kelve, M (1995) The (2¢-5¢) oligoadenylate synthetase is present in the lowest multicellular organisms, the marine sponges: demonstration of the existence and identification of its reaction products Eur J Biochem 232, 351–357.
31 Kuusksalu, A., Subbi, J., Pehk, T., Reintamm, T., Mu¨ller, W.E.G.
& Kelve, M (1998) (2¢-5¢) Oligoadenylate synthetase in marine sponges: Identification of its reaction products Eur J Biochem.
257, 420–426.
32 Wiens, M., Kuusksalu, A., Kelve, M & Mu¨ller, W.E.G (1999) Origin of the interferon-inducible (2¢-5¢) oligoadenylate synthe-tases: cloning of the (2¢-5¢) oligoadenylate synthetase from the marine sponge Geodia cydonium FEBS Lett 462, 12–18.
33 Custodio, M.R., Prokic, I., Steffen, R., Koziol, C., Borojevic, R., Bru¨mmer, F., Nickel, M & Mu¨ller, W.E.G (1998) Primmorphs generated from dissociated cells of the sponge Suberites domun-cula: a model system for studies of cell proliferation and cell death Mech Ageing Dev 105, 45–59.
34 Mu¨ller, W.E.G., Wiens, M., Batel, R., Steffen, R., Borojevic, R & Custodio, M.R (1999) Establishment of a primary cell culture from a sponge: primmorphs from Suberites domuncula Mar Ecol Progr Ser 178, 205–219.
35 Ulevitch, R.J & Tobias, P.S (1994) Recognition of endotoxin by cells leading to transmembrane signaling Curr Opin Immunol 6, 125–130.
36 Rottmann, M., Schro¨der, H.C., Gramzow, M., Renneisen, K., Kurelec, B., Dorn, A., Friese, U & Mu¨ller, W.E.G (1987) Specific phosphorylation of proteins in pore complex-laminae from the sponge Geodia cydonium by the homologous aggregation factor and phorbol ester Role of protein kinase C in the phosphorylation
of DNA topoisomerase II EMBO J 6, 3939–3944.
37 Krasko, A., Batel, R., Schro¨der, H.C., Mu¨ller, I.M & Mu¨ller, W.E.G (2000) Expression of silicatein and collagen genes in the marine sponge Suberites domuncula is controlled by silicate and myotrophin Europ J Biochem 267, 4878–4887.
38 Hovanessian, A.G., Brown, R.E., Martin, E.M., Roberts, W.K., Knight, M & Kerr, I.M (1981) Enzymic synthesis, purification, and fractionation of (2¢-5¢)-oligoadenylic acid Meth Enzymol 79, 184–193.
39 Brown, R.E., Cayley, P.J & Kerr, I.A (1981) Analysis of (2¢-5¢)-oligo (A) and related (2¢-5¢)-oligonucleotides by high-performance liquid chromatograpy Methods Enzymol 79, 208–216.
40 Kruse, M., Mu¨ller, I.M & Mu¨ller, W.E.G (1997) Early evolution
of metazoan serine/threonine and tyrosine kinases: identification