Clin. e.xp. Immunol.

(1989) 75, 336-342

Antibody binding of macromolecular DNA and RNA in the plasma of

SLE patients

F. KRAPF. M. H E R R M A N N , W. LEITMANN & J. R. KALDEN Institute for Clinical Immunology and

Rheumatology. University of Ertangen-NUrnherg, Krankenhausstr. 12. D-8520 Erlangen, FRG

( Accepted for puhlication 21 September 19H8)

SUMMARY
Plasmapheresis fltiids from 20 palients with clinically active SLE. from three patients with
Waldenstrom's disease, from three patients with rheumatoid arthritis, two patients with myasthenia
gravis and other diseases including aetive systemic disorders were precipitated using polyethylene
glycol 6000 (PEG). By applying ethidium bromide staining, plasma nucleic acids (PNA) could be
demonstrated in PEG-precipitates of SLE patients exclusively. Purified immunoglobuhns of SLE
plasma precipitates were shown to form antigen-antibody complexes with PNA as dcmonstraied by
eleclronmicroscopy. Further charactcrizalion o^ PNA by agarose gel eleetrophoresis revealed a
molecular weight up to 20 kbp. Cesium chloride buoyant density gradients showed non-
homogeneous molecules, excluding pure microbial origin. In spite of RNase digestion, the PNA
contained RNA with 30-70"ii riboguanosine as shown by nucleoside analysis. The high amount of
guanosine-rich RNA was further supported by similarities between PNA and polyriboguanylic acid
in hyperehrome shifting due to thermic denaturation. HPLC analysis showed a molecular weight of
ribonucleic acids of more than 60 b thus excluding mere oligonueleotides. In contrast to B-type
dsDNA. PNA from SLE patients were immunogenic. Antibodies against PNA could be induced in
rabbits by subcutaneous injection. The anliscra thus obtained showed crossrcactivity with
polyriboguanylic acid and dsDNA preparations.

Keywords nucleic acids SLE plasma

INTRODUCTION presence ofimmunoglobulin-bound double helical NA (Krapf,

Circulating nucleic acids (NA) in sera or plasma of patients
suffering from systemic lupus erythematosus (SLE) have been
repeatedly described (ikcbe, Gupta & Tan. 1983; McCoubrey-
Hoyer, Okarma&Holman I9S4; van Helden, 1985). The size of
the.se NA as reported by different authors ranges between 20 bp
(Papaliancr^/.- 1980) and 17-5 kbp (Rieber £'/«/., 1986). Their
possible pathogenie role, however, is still unclear. It has still to
be discussed whether these DNA molecules are a specific finding
in SLE sera at all, or whelher they might be released following
cellular destruction during clotting processes (Steinman. 1975).
Moreover, the role of DNA-anti-DNA immune complexes
in the palhogenesis of SLE is still under discussion (Izui,
Lambert & Miescher. 1977; Davies. Godfrey & Wmfield, 1978;
Bruneau & Benvenisle. 1979; Manger ct ai 1985). In a previous
study we were able to demonstrate anti-immunoglobulin preci-
pitable material reacting with acridine orange, suggesting the
Correspondence: PD Dr K. Krapf, In.stitiile for Clinical Immu-
nology and Rheumatology, Universily Frlangen-Niirnberg. Kranken-
hausstr. 12, D-8520 Erlangen. KRG.
336
1984). However, sinee the method applied did not provide any
information aboul the physico-chemical properties of plasma
nucleic acids (PNA), we characterized the material further with
respect to structure, bioehemical properties and antigenieity.
Furthermore, the origin of PNA was investigated especially with
respect to the question of whether its existence may be due to
mere cellular destruction leading to unspecific NA release.
MATERIALS AND METHODS
Patients
Two litres of plasmapheresis fluid were obtained from each of 20
patients with clinically aetive SLE (Kalden 1985). EDTA and
NaNi (0-1 '/I.) were added immediately after plasma separation.
To avoid contamination with NA from cell detritus, the first 100
ml of the material was discarded. Identieal proeedures were
performed with patients suffering from Waldenstrom's disease
(/7 = 3), myasthenia gravis (« = 2), rheumatoid arthritis (/i = 3)
and, more recently, vasculitis (n = 2), Churg Strauss syndrome
(n= I), panniculitis («— I), and pancreatitis {n— I).
None of these patients nor plasma specimens from 100
healthy persons showed detectable amounts of double helical
 Antihoch-hound nucleic acids in SLE paiienis
NA, whereas plasmapheresis material from all 20 patients
suffering from clinically active SLE contained 30-400 ng/ml
dsDNA equivalent.
Preparation of prutein-htninil nucleic acids frotn plasniaplwresls
fluid
To discriminate between free and protein-bound DNA, aliquots
ofthe plasmapheresis fluid were incubated for 30 min with 5''n
PHCi60()0at4 C and centrifuged for 15 min at 10,000jf. Under
these conditions neither relevant amounts of free antibody
(Zubler et ai. 1977) nor free NA (Eshenbugh, Seno & James
1974). are precipitated, but protein bound NA eg. DNA-anti-
DNA immune complexes arc precipitated (Zubler et ai. 1977).
PEG-precipitates were redissolved in TES 100 (10 mM Tris, 1
mM EDTA, IOO min NaCl. pH 7'5) and divided into two
aliquots. One was used for the preparation of immunoglobulins
(Ig). the second for piirilication of NA. The latter was adjusted
to a conductivity of 20 mSicmens with 4 M NaCl and incubated
with 5 g DEAE cellulose/1. The gel was stored to settle, washed
three times with TES 100, and finally loaded onto a chromato-
graphy column. Elution of NA was performed by a LiCl-
gradient (1-4 M LiCl with ^-S'Vu lithiumdodccylsulphate). The
eluate was extracted with TES IOO saturated phenol, twice with
chloroform isoamyl alcohol (24: I), finally with ether, and then
precipitated with 2 5 vol. of ethanol for 20 h at —20 C. The
precipitate was washed twice with 70".. ethanol. redissolved in I
ml distilled water, treated with 50//g/ml RNaseAat 37 C for 30
min. diluted with 5 ml TES 100 plus 1% SDS and digested for2h
at 37 C with 50 /ig/ml proteinase K. The material was rc-
extracted using TES 100 saturated phenol, followed by chloro-
form isoamyl alcohol (24:1) and precipitated with 2-5 vol.
ethanol at —20 C. samples were redissolved in distilled water.
Quantification ofthc prepared NA and tests of purity were
performed using a UV absorption spectrum between 220 and
300 nm and cthidium bromide fluorescence.
PECi-prccipitabIc immunoglohulins
Eor preparation of Ig, redissolved PEG-precipitated plasma-
pheresis material was incubated with 0 5 vol. of saturated
ammonium sulphate for 10 min at 4 C, followed by centrifuga-
tion for 10 min (4 C, 2.000 A'). The precipitate was redissolved in
O-I vol. of distilled water. This procedure was repeated twice.
Then the Ig solution was dialyzed against 20 mM Na.iP04. After
absorption with DEAE cellulose the unbound proteins were
analyzed by means ofan HPLC system (LKB, Sweden).
ELISA for the detection af antibodies against NA in rabbits
Microtitre plates (NUNC. Denmark) were coated with 20/(g/ml
Poly-L-lysine (Sigma. USA) in TE (20 mM Tris, 5 mM EDTA. pf 1
7 5). After incubation for 2 h at 37 C. the plates were washed
with TE and coated with PNA. polyriboguanylic acid (pG.
Sigma), or dsDNA (Sigma) for 16 h at 4 C at concentrations of
20 /(g/ml in TE. Coating efficiency was about 10'"... Double
stranded DNA was prepared irom calf thymus DNA by
nuclease SI digestion and subsequent chromatography on
BND-cclluIose (BioRad. USA) (Kiger & Sinsheimer. 1969).
Test sera were diluted I/IO. 1/20. 1/40 up to 1/2560 in
phosphate-buffered saline containing 0-05'>;> Tween 20 and
incubated for 2 h at 37 C. Detection of bound antibodies was
achieved by horseradish pcroxidase-coupled goat anti-human
337
IgG antibody (Tago. USA). After 30 min incubation, the
dilution which yielded an increase in optical density (OD) ofO 5
was regarded as the dsDNA-antibody titre of the sample.
Immunization of animals
Seven 6-month old New Zealand red rabbits were immunized
with 50 j.1% ofdsDNA, pG and PNA from SLE patients with or
without methylated BSA (mBSA) carrier, respectively, in 1 ml
Freund's complete adjuvant (KCA) by five intramuscular
injections at weekly intervals: mBSA in I ml ECA served as
control.
Gel electrophoresis
Gel electrophoresis in 1 % agarose gels was performed in 50 mM
Tris acetate. pH l-'i. Hind III cut phagc lambda DNA and Hae
III cut replicativc form of phage 0X174 DNA served as
markers.
Melting curves (if nucleic acids
Temperature-dependent hyperchromc shifting of the NA (50-
100 ng/ml) was measured in 0-042 M sodium phosphate. pH 6'8.
using a spectrophotometer with a heated cuvette (Gilford.
USA). The melting curve was registered at 254 nm with
automatically performed zero adjustment.
CsCI buoyant density
The CsCl buoyant density gradient was performed using an
analytic ultracentrifuge (Beekmann, FRG) at 44.000 rpm for
48 h (initial CsCl density: 1 -7 /ig/ml). For standardization, DNA
from E. Coli with known density was used. NA were detected
optically at 260 nm during the run.
Hybridization
Purified NA were spotted on a nylon membrane (Zeta Probe.
BioRad) and dried for 6 hat 80 C. The hybridization probe used
was "-P-labelled BLUR 12 DNA (lO*" ct/min//ig). recognizing
highly repetitive Alu-sequences of the human genome (Dein-
inger et al. 1981). Hybridization was performed in 5O> ' ^>
formamide at 42 C for 24 h. Then the filters were washed at
65 C with SSC (15 mM sodium citrate. 150 niM NaCl. pH 7-2),
0-2 X SSC, and 0' i x SSC. dried and autoradiographed.
Nucleoside analysis
The NA preparations were digested with DNase I and nuclease
PI, and the resulting nucleolides were dephosphorylated with
bacterial alkaline phosphatase (Kuo et ai. 1981). Nucleoside
composition was investigated by HPLC analysis (The Supelco
Reporter, 1985), and quantification was performed measuring
optical density at 280 nm.
F.lcctronmicro,scopy
For electronmicroscopy NA were dissolved in buffered forma-
mide (5O';^>) including cytochromc C and spread on formamide
(30"/;i). The layer containing NA was picked up using a
collodion-covered copper net. fixed in acidic-alcoholic uranyl
acetate, dehydrated with increasing concentrations of ethanol
and covered with platinum. For demonstration of antigen-
antibody complexes, the isolated PNA and Ig prepared ofthe
same plasmapheresis fluid were incubated, and the reformed
immune complexes suspended in TES 100 were absorbed to
freshly split glimmer. After fixation they were vaporized with
 338 F. Krapf el al.
carbon. Glimmer platelets were washed and finally pieked up as 0 % SC
described above. The lenglh of the NA was measured using an
optometrical scanner. A copper net with known dimensions
served as length standard.

RESULTS

Preparation of PNA and Ig

All patients (n = 20) suffering from clinically active SLE showed
PNA eoneentrations between 30 and 400 ng/ml dsDNA equiva-
lent. No dsDNA could be demonstrated in plasmapheresis
fluids of three patients suffering from Waldenstrom's disease,
three patients with rheumatoid arthritis, two patients with
myasthenia gravis. and all other patients as indieated above.
The purified PEG-preeipitible Ig from plasmapheresis fluids
of SLE patients contained 90'^ IgG. 5% IgM, and 1 2%
transferrin and albumin, respectively (data not shown).

Characterization of PNA
Negative hybridization with BLUR 12 (Bam H I-Linked
Unique Repeat) DNA, recognizing highly dispersed alu
sequences, exeluded the possible origin ofPNA from unselected
human DNA (data not shown). In addition, free haemoglobin
was determined to be less than 5 /Jg/ml in all cases studied,
indieating no relevant cell rupture during plasmapheresis (less
than 0-01 "-iO.
Applying CsCl buoyant density eentrifugation. a non-
homogeneous density of PNA was demonstrated, indicating a
high complexity of PNA (Fig. I).
Gel eleetrophoresis showed PNA of non-homogeneous Fig. I. An analytic cesium chloride buoyant density gradient of PNA
molecular weights. However, the presenee of high molecular from one patient with SLE as compared to a nucieic acid preparation of
weight NA comigrating with 20 kbp linear dsDNA was E. coli obtained under same conditions.
demonstrated in most ofthe preparations investigated (Eig. 2).
Nucleoside analysis after complete DNase 1. PI nuclease,
and alkaline phosphatase digestion showed an elevated dG-dC
eontent as eompared to random human genomic and mitoehon-
drial DNA (Table 1) of hepatoeytes.
lo lb 2a 2b 3o 30 4c 4b 5a 5b W 6a I I
Poor methylation of deoxyeytosine, about I % as compared
to 2-5-4-5'Mi of human eellular DNA, eould be proven by
nueleoside analysis (data not shown).
In spite of RNase A digestion during the preparation of
rtfl 23 I kbp
9 4 kbp
PNA, high amounts of ribonucleosides. espeeially riboguano- 6 6 kbp

sine, were evident as shown in Table 2.

4 4 kbp
In further HPLC analyses, the absence of oligoribonueleo-
tides ai' less than 60 b was proven (data not shown). These
findings exclude the possibility that the RNA contents of PNA
were due to eo-preeipitated oligoribonueleotides. * 2 3 kbp
2 0 kbp
Measurement of hyperchrome shifting during thermal dena-
turation showed an elevated melting point of all PNA as
eompared to random human DNA. Melting eurves of PNA 564 bp
showed a minimal optieal density at 40 C followed by a steady
increase without any obvious transition point up to IOO C.
i 125 bp

Upon cooling, ihe obtained re-assoeiation pattern differed

significantly from common B-DNA since OD values of the
samples deereased rapidly to a minimum at 40 C and increased
slightly between 40 C and 25 C. In eontrast, random cellular
dsDNA showed a continuous deeline of OD down to 25 C. A
typical melting eitrve of PNA as cornpared to human eellular
DNA and pG is given in Eig. 3, demonstrating similarities
between pG and PNA from SLE patients.
Fig. 2, Agarose gel electrophoresis of PEG-precJpitible nucleic acid
fractions of several palienls with and without DNase treatment in
comparison to nucleic acids of two human B cell lines prepared under
the same conditions, and to lambda phage DNA, la 7a Nucleic acid
fractions of palients 1-7,1 b-5b DNase trealed nucleic acid fractions of
patients 1-5, M Marker lambda, Hind III digested, I, II Nucleic acid
fractions ofhuman B cell lines (analogously prepared).
 Antibody-hound nucleic acids in SLE patients 339
Table I. DN.A nucleoside composition in PNA isolated from SLE patients"
plasma

Deoxyribonucleosides (mol %)

Patient dA dC* dCi dT dG + dC Purines dG/dC dA/dT

EM 18'0 30'0 3i'O 20-0 61 49 1-03 0-90

FH 21-0 29-0 26-0 23-0 55 47 0'90 091
HI 19-0 29-0 32-0 200 61 51 1-10 0-95
KA 20 0 290 26-0 25-0 55 46 0-90 0-80
ME 24-0 28-0 26-0 22-0 54 50 0 93 1 09
MJ 22'0 29'0 25'0 23-0 54 47 0S6 0-96
SW 24'5 28-5 24-5 22-5 53 49 0-86 1-09
SLF.t 22 29 26 22 55 48 0-90 1-00

human D N A
nuclear 29 21-4 21-2 28-4 42-6 50-2 0'99 I 02
t milochondrial 27-8 2 2 1 2 2 1 27-8 44-3 50-0 1-00 ! 00

* dC plus m-dC
t Pooled fraction.
JAndersoncf a/. (1981).

Table 2. Nucleoside composition of RNA isolated from plasma

of SLE patients with and without RNase A treatment

Riboiiucleosides (mol "A ,)

Patient rA rC rG rU Purines

FM 20 30 34 16 54
FH 23-5 23-5 37-5 155 61
HH* 17 16 55 12 72
HI 26 19-5 40-5 14 66-5
KA* 17 18 53 12 70 Q
dto* 23 20 43 14 66 O

MF 17-5 17-5 53 12 70'5

MJ 2 25 35 IS 57
SW* 25 18 45 12 70
SLEt \X II 63 S SI

No RNA could be demonstrated in other patients with

chronic inflammatory diseases and healthy controls.
* Trented during the preparation with RNase A. 0-8 -
+ Pool of several SLE patients, further purified for removal of
oligonucleotides.
30 40 50 60 70 80 25
Temperature (°C)
Electron microscopy of the PNA did show not only dsDNA
including conformations resembling hairpins and strandcrosses. Fig. 3. Hypcrchrome shifting of optical density of PNA ( ),
but also a small amount (-^3'/'^) of unusually structured polyriboguanylic acid (- • - • - ) . and dsDN A due lo thermic denaturalion
molecules without the typical characteristics of DNA supercoils ( ). Similarities could be demonstrated between PNA and polyri-
(not shown). The length of the NA was up to 7 nm. thus boguanylic acid, but not with ds-DNA.
confirming the estimation ofthcsizeof 20kbp. as determined by
gel electrophoresis.
vitro formation of PNA and dsDNA immune complexes could
Formation of in vitro immune complexes be confirmed by electronmicroscopy. In addition it couid be
The mere possibility of precipitating NA by applying PEG 6000 demonstrated that antibodies prepared from SLE plasmaphere-
at a final concentration of 5'^^ does not prove the existence of sis fluid react preferably with hairpins, strand crosses and other
DNA-anli-DNA complexes. Therefore it was of interest to test uncommon structures ofthc corresponding NA (Fig. 4). Yet it
whether the antibodies of SLE patients could react in ritro with cannoi be excluded that bivaleni anlibodies may account for the
PNA obtained from the same sample of SLE plasma. The in- observed structures by cross-linking phenomena.
340 F. Krapfet al.

Fig. 4. Electronmicroscopy of the in-vitro formation oT antigen-antihody complexes consisting of PEG-precipitib!e PNA and
immunoglobulins from SLE patients. Note that the interaction between the antibodies and the nucleic acids is focused on strand crosses
or links.

Table i. Antibody titres against dsDNA and pG in rabhits after

immunization with nucleic acids

dsDNA pG
Days after immunization Days after immunization

0 21 35 60 67 Injected NA 0 21 35 60 67

10 10 10 10 10 salmon testis 10 40 40 10 10
10 10 40 20 10 dto. plus mBSA 10 40 40 160 320
10 10 10 80 160 pG 10 10 160 160 160
10 10 10 640 1280 dto. plus mBSA !0 320 160 1280 1280
10 10 160 320 320 SLE-PNA [0 10 SO 320 320
10 160 20 40 40 dto. plus mBSA 10 10 10 10 10
10 10 io 10 20 mBSA (control) 10 10 10 10 10

The titre represents the dilution of scrum which yielded an increase in

OD vLtlues of 0 5 within 30 min in an ELISA.

Immunogenicity of PNA in animals
Since common B-DNA has no immunogeneic potency (Stollar,
1986). the capacity o\' PNA from SLE patients to induce
antibody production in animals was investigated. For this
purpose PNA. dsDNA and pG were injected into rabbits. As
shown in Table 3. PNA-induccd antisera reacted not only with
PNA, but also with dsDNA and pG. Similar results were
obtained using pG as immunizing agent, whereas it was not
possible to induce such antibody specificities by the application
ofdsDNA(Tablc3).
A fluid phase inhibition ofanti-dsDNA titres of 80% could
be achieved by pre-incubation ofthc antisera with 0-1-10/<g calf
thymus DNA, corresponding to 10 '^-10 •* M dsDNA (not
shown). Using the described ELISAassay, it was not possible to
directly compare rabbit anti-sera titres with those obtained from
actual SLE patients. A similar assay applied for human anti-
DNA antibody detection showed anti-dsDNA titres ranging
from l/lOO- l/IO** in SLE patients with identical estimation of
OD values, suggesting comparable amounts of anti-DNA
activity (data not shown). However, the application of a
 Anlihody-bound nucleic acids in SLE patients
commercially available FARR assay system (Amersham) failed
lo detect DNA precipitating activity of rabbit antibodies, thus
indicating a lower allinity of polyclonal rabbit antisera as
compared to some of the sera derived from patients with
clinically active SLE.
DISCUSSION
Antibodies against dsDNA and/or ssDNA are a characteristic
finding in sera of patients suffering frotn SLE. Steinman and
Ackad (1977) suggested that the amount of DNA in the plasma
correlated directly wilh the severity of the disease and especially
to vasculitic symptoms. A diminished activity of plasma
nucleases corresponding to disease activity was suggested to be
responsible for this finding (Chitrabamrung. Rubin & Tan.,
1984). Upon involvement of glomerulonephritis in advanced
stages of the disease, the formation and deposition of DNA-
anli-DNA immune complexes appears to be crucial.
The demonstration of uncommon biological properties or
PNA of SLE patients was primarily attributed to the molecular
weight. The steady increase of molecular weight of the detected
NA described during the past years by several authors (Sano &
Morimoto, 1982); McCoubrcy-Hoyer er «/., 1984; van Helden,
1985: Rieber ei at. 1986) is possibly due to methodological
iinprovements. However, high amounts of dG-dC base pairs
and potential Z-DNA forming regions in the isolated NA
molecules were reported also (van Helden, 1985).
The existence of DNA-anti-DNA immune complexes in
plasmapheresis fluids of SLE patients, in contrast to all controls,
could be demonstrated by rc-association experiments applying
purified NA and Ig. We detected measurable amounts of NA
(> 20 ng/ml) exclusively in plasmapheresis fluids of SLE
patients, whereas this could not be shown even m severe cases of
other systemic disorders. As demonstrated by agarose gel
electrophoresis in the present study, high molecular weight NA
of up to 20 kbp could be detected in all cases of SLE.
The dG-dC content of PNA was up to 61";) as compared to
.^S"u in bulk human DNA. Although the presence of Z-DNA in
ihe PNA cannot totally be excluded, the in-vivo stabilization of
Z-DNA is thought to require a high degree of cytosinc
iiiethylation. and, therefore, the poor content of mMC in PNA
makes relevant amounts of Z-DNA unlikely. The dsDNA
exhibited nicks, hairpins, and other uncommon structures,
which may serve as targets of antibodies in SLE patients" serum
(Reichlin & Mattioli, 1973, 1974): Stollar 1986). The CsCI
buoyant density gradient showed heterogeneous NA, excluding
mere bacterial origin. Furthermore, the melting curves of NA
showed similarities to those of pG suggesting a high amount of
guanosine-rich RNA in spite of RNase digestion during the
purification procedure. The high content of RNA was further
substantiated by nucleoside analysis. High amounts of RNA
with guanosine contents from 30-10% could be demonstrated
(Table 2), thus supporting the findings obtained by the thermal
denaturation studies.
Since it was shown before that B-DNA is not immunogeneic
(Stollar, 1986). it was crucial to prove the immunogeneic
properties of PNA isolated from SLE patients. Rabbits iinmu-
nizcd with PNA or pG developed antibodies reactive with PNA,
pG and dsDNA. consistent with the data reeently published
(Eilat & l.otan. 1982: Eilat ei ai. 1984). These results raise the
possibility that uncommon DNA and/or RNA structures might
341
induce antibodies cross-reaetive with dsDNA. Indeed, the
presence of mBSA carrier in the immunization mixtures con-
taining PNA abolished the immunogenic potency of DNA
(Tabte 3) almost completely, indicating that conformational
rather than linear nucleotide antigenic determinants are crucial
for antigenicily. In contrast, animals immunized with dsDNA
did not produce antibodies against NA. A transient low anti-
DNA antibody titre was probably due to a polyclonal B cetl
response to Freund's adjuvant. These data strongly indicate that
RNA or DNA/RNA mixtures induce antibodies cross-reacting
with dsDNA. which might compete with plasma nucleases and
inhibit the enzymatic digestion of circulating DNA in the scrum
(Chitrabamrung el ai. 1984). Further investigations will be
necessary to determine if this phenomenon is responsible for the
induction of polynuelcotide antibodies found in SLE patients.
A possible origin of plasma DNA could be undermethy-
lated, GpC-rich 'HTF-islands' in the human genome, as
reported by Bird (1986), since these islets have similar features to
those demonstrated in the plasma DNA in the present investiga-
tion. The origin of RNase-resistant purine-rich RNA with more
than 60 b is unknown, but long purine stretches are frequently
observed in unusually structured RNA, eg. rRNA. Yet this
presumption needs experimental support.
ACKNOWLEDGMENTS
This work was supported hy the Deutsche Forschungsgemeinschaft.
Grants SFB 118 Bl and Kr 798.1-1. The editorial help of Mrs U. Rath
and Mrs L. Kaunt? is gratefully acknowledged by t!ie authors.
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