Anti-RNA polymerase I antibodies in sera of MRL lpr/lpr and MRL +/+ autoimmune mice. Correlation of antibody production with delayed onset of lupus-like disease in MRL +/+ mice.

Sera from individual MRL/lpr and MRL/++ mice, which develop an autoimmune disease similar to human systemic lupus erythematosus (SLE), were screened over a period of approximately 30 wk for the presence of anti-RNA polymerase I and anti-ssDNA antibodies. Even though onset of the disease is delayed in MRL/++ as compared to MRL/lpr mice, anti-ssDNA antibodies were present in comparable concentrations in the sera of all mice by the age of 6 wk. As observed in sera of human SLE patients, anti-RNA polymerase I antibodies were detected in the sera of all MRL mice. However, unlike the anti-ssDNA antibodies, anti-RNA polymerase I antibodies were detected much later in MRL/++ mice (mean age, 22.8 wk) as compared to MRL/lpr mice (mean age, 9.6 wk). The presence of anti-RNA polymerase I antibodies in sera of MRL mice was thus a much better indicator of disease status than the presence of anti-ssDNA antibodies. The appearance and increase in anti-RNA polymerase I antibodies in the sera of MRL/++ mice correlated (R2 = 0.964) with a precipitous decrease in anti-ssDNA antibodies, starting at about 20 wk of age. These results suggest a possible relationship between the RNA polymerase I and DNA autoimmune reactions.

Microbodies in germinating fern spores: evidence for glyoxysomal activity.

Enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, are active during the germination of spores of the fern Dryopteris filix-mas. Increases in activity of both enzymes are correlated with the breakdown of lipid reserves. The occurrence of these enzymes suggests that the microbodies previously described in these spores are glyoxysomes.

Segregation and mapping analysis of polymorphic HLA class I restriction fragments: detection of a novel fragment.

An HLA-B7 complementary DNA clone was used as a hybridization probe to analyze the segregation pattern of polymorphic class I restriction fragments in several families whose HLA types had been determined by serological techniques. In one family in which a crossover in the HLA region had occurred, a specific genomic fragment was mapped with respect to the crossover. In another family, a novel genomic fragment present in one child and absent in all other family members was observed. With the exception of this novel fragment, all polymorphic class I fragments observed in this study segregated with a serologically defined parental haplotype, a result consistent with HLA linkage.

Protein kinase NII. Interaction with RNA polymerase II and contribution to immunological cross-reactivity of RNA polymerases I and II.

The interaction between antibodies directed against RNA polymerase I purified from Morris hepatoma 3924A and homologous RNA polymerase II was investigated. The activity of partially purified polymerase II was inhibited by the antibodies. In contrast, the reaction catalyzed by the purified enzyme was not affected. Partially purified polymerase II preparations contained a protein kinase activity. Sucrose gradient centrifugation in the presence of 0.3 M KCl resulted in complete separation of RNA polymerase II from protein kinase as well as in complete loss of sensitivity to the anti-RNA polymerase I antibodies. The protein kinase possessed reaction characteristics similar to those of the NII protein kinase (Rose, K.M., Bell, L.E., Siefken, D.A. and Jacob, S.T. (1981) J. Biol. Chem. 256, 7468-7477) which is associated with hepatoma RNA polymerase I (Rose, K.M., Stetler, D.A. and Jacob, S.T. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2833-2837). The activities of both kinases were inhibited to the same extent by anti-RNA polymerase I antibodies and polypeptides of Mr 42 000 and 25 000, present in both kinase preparations, formed immune complexes with the antisera. Readdition of protein kinase NII to purified polymerase II resulted in phosphorylation of the polymerase and a concomitant enhancement of RNA synthesis. After addition of the kinase, RNA polymerase II activity was again sensitive to anti-RNA polymerase I antibodies. Upon reacting with protein kinase NII, RNA polymerase II polypeptides could be detected in immune complexes with anti-RNA polymerase I antibodies. These data indicate that protein kinase NII is associated with RNA polymerase II during early stages of purification and is at least partially responsible for the immunological cross-reactivity of RNA polymerases I and II.

Characterization of a yeast phase specific protein from a fungus, Histoplasma capsulatum.

We have characterized an unusual yeast phase specific protein from Histoplasma capsulatum. The protein, which we have called protein 6, is produced by the yeast cells which have been derepressed for sulfite reductase, and it can account for more than 40% of the total extract protein. Synthesis of both sulfite reductase and protein 6 is subject to cysteine repression. However, sulfite reductase activity is maximal in logarithmically growing cells whereas protein 6 is synthesized de novo and accumulated by stationary phase cells. The following are the major physicochemical properties of protein 6: (1) the native protein has a molecular weight of about 15 000; (2) electrophoresis on a sodium dodecyl sulfate polyacrylamide gel yielded a single band with a molecular weight 7600; (3) protein 6 is capable of reducing the dye, nitroblue tetrazolium, and cytochrome c, a property that has been found to be shared by a number of trypsin inhibitors, and (4) the molecule is negatively charge and is relatively resistant to proteolysis. The amino acid composition of protein 6 has been determined.

Expression of Sporophytic Storage Proteins in the Corm of the Quillwort (Isoetes echinospora Dur.).

Parenchyma cells from the corm tissue of the aquatic lycopod Isoetes echinospora Dur. were shown by electron microscopy to be packed with amyloplasts, lipid bodies, and protein bodies. The protein bodies are morphologically similar to those identified in seeds and certain vegetative tissues of higher plants. Globoid-containing protein bodies (1-10 [mu]m) isolated in a sucrose gradient possessed a buoyant density of 1.28 g/mL and contained globulin (salt-soluble) proteins. Sucrose gradient centrifugation of crude globulins revealed only two components with mean sedimentation coefficients of approximately 2S and 11S. The 2S component, designated VSP-IsA, was composed of a 15.7-kD polypeptide. The 11S component, designated VSP-IsB, had a molecular mass of 215 kD as estimated by gel filtration and was composed of 39- to 42-kD polypeptides. Two-dimensional gel electrophoresis showed constituent polypeptides distinguished by differences in net charge and molecular mass. Affinity-purified antibodies against VSP-IsA and VSP-IsB prepared and used as probes on immunoblots cross-react only with their specific antigens, suggesting that the proteins are not immunologically related. Indirect immunolocalization studies confirmed that VSP-IsB is deposited in protein bodies. These globulin proteins, like those from some seeds, form the principal storage reserves of the corm tissue.

Multiple GABAA receptor alpha subunit mRNAs revealed by developmental and regional expression in rat, chicken and human brain.

GABAA receptor alpha subunit transcripts were detected by Northern analysis of rat, chicken and human brain mRNA using a series of 32P-labelled antisense RNA probes derived from human alpha 1 subunit cDNAs. These alpha subunit mRNAs differ in their distribution among various brain regions in the rat and at least one species is detected primarily in fetal brain. GABAA receptor alpha 1 subunit probes encoding the putative extracellular domain detect at least five alpha subunit transcripts in rat brain, whereas probes encoding the putative intracellular domain detect only two mRNAs. These data suggest the presence in brain of multiple GABAA receptor alpha subunits having homologous extracellular domains and whose expression is regionally and developmentally regulated. These alpha subunit transcripts may encode proteins that comprise GABAA isoreceptors differing in their pharmacological and physiological properties.

Structural comparison of the Autographa californica nuclear polyhedrosis virus-induced RNA polymerase and the three nuclear RNA polymerases from the host, Spodoptera frugiperda.

A partial subunit structure has been determined for the novel RNA polymerase that is induced in fall armyworm (Spodoptera frugiperda) cells upon infection with the Autographa californica nuclear polyhedrosis virus (AcNPV). The putative structure includes nine polypeptides; the complexity of this structure is in accord with the high sedimentation coefficient (15S) estimated for this enzyme. A comparison of the putative structure of the virus-induced polymerase with those of the three host nuclear RNA polymerases shows that the structure of the viral polymerase is apparently unlike any of the host nuclear polymerases. This conclusion is reinforced by immunoblot experiments that show no cross-reactivity between the virus-induced polymerase and an antiserum directed against Drosophila RNA polymerase II. The virus-induced RNA polymerase appears at the onset of the late phase of infection and still appears when viral DNA synthesis is blocked by aphidicolin. Thus, the virus-induced polymerase seems to be composed of early viral products.

Monoclonal antibody against the lupus antigen Sm cross-reacts with RNA polymerase I.

Monoclonal anti-Sm antibody, a specificity directed against a constituent of nuclear ribonucleoprotein and considered to be a marker for systemic lupus erythematosus (SLE), was tested for its ability to react with four other rheumatic disease antigens of known enzymatic activity. No binding of the antibody was observed in radioimmunoassays with immobilized protein kinase NII, poly(A) polymerase, or topoisomerase I. In contrast, anti-Sm antibody did react with RNA polymerase I. Under conditions of antibody excess, anti-Sm was determined to bind RNA polymerase I on an equimolar basis, indicating that the polymerase possesses a single epitope recognized by the anti-Sm antibody. Addition of the anti-Sm antibody to in vitro transcription reactions resulted in inhibition of RNA polymerase I activity but had no effect on the reaction catalyzed by RNA polymerase II. When the subunits of RNA polymerase I were separated by polyacrylamide gel electrophoresis under denaturing conditions and incorporated individually into the radioimmunoassay, anti-Sm antibody bound only to the sixth polymerase polypeptide (Mr, 21,000). These data establish an immunological relationship between two important rheumatic disease antigens and help explain the apparent diversity of the autoimmune response in murine and human SLE.

Rabbits produce SLE-like anti-RNA polymerase I and anti-DNA autoantibodies in responses to immunization with either human or murine SLE anti-DNA antibodies.

Anti-DNA and anti-DNA polymerase I (RPI) autoantibody responses are symptoms of systemic lupus erythematosus (SLE). To investigate the relationship between these antibodies (Ab), rabbits were immunized with one of the following preparations: human SLE anti-DNA Ab; human SLE anti-DNA IgG; normal human anti-DNA Ab; human Grave's disease anti-DNA Ab; murine SLE anti-DNA Ab or anti-DNA IgG Fab; various normal human, murine, or rabbit IgG preparations; or complete Freund's adjuvant (CFA), alone. All of the animals immunized with anti-DNA Ab (n = 14) generated Ab reactive in radioimmunoassay with: ssDNA, dsDNA, RPI, the soluble fraction of rabbit liver crude nuclear extract, and the immunogen. Induced rabbit anti-DNA Ab in turn induced these responses in a different rabbit: a rabbit immunized with rabbit anti-DNA IgG Ab which had been previously induced by immunization with human anti-DNA Ab, produced Ab reactive with ssDNA, dsDNA, RPI, and the soluble fraction of rabbit liver nuclear extract. Although an individual animal's antisera reacted consistently over the course of immunization with the same individual RPI subunit(s), antisera from different animals reacted with different subunits of the 9-subunit RPI complex in Western blot analyses: 190 kD (n = 6); 120 kD (n = 1); 62 kD (n = 4); 45 kD (n = 2); and, no reactivity (n = 2). In contrast, animals immunized with normal IgG or CFA produced responses only against the immunogen. Together, these data suggest that anti-DNA and anti-RPI responses are connected through an autoimmune network in SLE.

Anti-RNA polymerase I antibodies in the sera of MRL lupus mice at the initial stages of disease are directed primarily against phosphorylation-dependent epitopes.

Anti-RNA polymerase I (RPI) antibodies in the sera of MRL/Mp-lpr/lpr and MRL/Mp(-)+/+ mice, which develop an autoimmune disease similar to human systemic lupus erythematosus, were screened for reactivity with purified RPI or RPI which had been dephosphorylated. In every case (n = 10), dephosphorylation of RPI resulted in a significant decrease (33-95%) in antibody binding. The anti-RPI antibodies in the sera of the same mice approximately 6 weeks later also reacted better with untreated as compared to dephosphorylated RPI but, in every case, the decrease in antibody (0-30%) caused by dephosphorylation was substantially diminished. That the proportion of anti-RPI antibodies in the sera of MRL mice decreased with progression of lupus-like disease was confirmed by closely monitoring the antibodies over the course of disease. Anti-RPI antibodies produced at the earliest stages appeared to be directed almost exclusively against phosphorylation-dependent determinants since dephosphorylation of RPI essentially abolished antibody binding. Subsequently, the percentage of the total anti-RPI antibodies in the sera of these mice directed towards phosphorylation-independent epitopes increased linearly with time. The importance of phosphorylation-dependent epitopes on RPI for the development of the anti-RPI autoimmune response was supported by the observation that treatment of mice with alkaline phosphatase partially attenuated anti-RPI antibody production.

RNA polymerase I in hepatoma 3924A: mechanism of enhanced activity relative to liver.

Two major cyclic nucleotide-independent protein kinases, NI and NII, have been identified in Morris hepatoma 3924A and rat liver. When expressed per unit DNA, the activities of protein kinase NI and NII were 1.3 and 12 times greater, respectively, in the hepatoma than in liver. Protein kinase NII, but not NI, was capable of phosphorylating and activating the DNA-dependent RNA polymerases I and II. Phosphorylation of RNA polymerase I was accompanied by an increase in average size of the RNA synthesized in vitro, whereas phosphorylation of RNA polymerase II was concomitant with an elevation in the number of RNA chains initiated. RNA polymerase I polypeptides of Mr 120,000, 65,000 and 25,000 were phosphorylated by protein kinase NII; RNA polymerase II polypeptides of Mr 214,000, 140,000 and 21,000 were modified by this kinase. In contrast to the purified hepatoma enzyme, RNA polymerase I activity in nuclear lysates was not affected by addition of protein kinase NII. In vitro phosphorylation of the tumor lysate followed by immunoprecipitation of RNA polymerase I polypeptides indicated little or no phosphate transfer to the 65,000 Mr polypeptide of the enzyme. These data suggested that the tumor enzyme, particularly the 65,000 Mr polypeptide, was highly phosphorylated in vivo, but becomes dephosphorylated during purification. Unlike the tumor enzyme, RNA polymerase I in the liver lysate responded to protein kinase addition; phosphorylation of the liver polymerase I polypeptides of Mr 120,000, 65,000 and 25,000 was observed. These observations indicate that the liver enzyme is not completely phosphorylated (activated) in vivo and that the relatively rapid rate of ribosomal RNA synthesis in the rapidly growing hepatoma may result, at least in part, from a polymerase I which is maximally phosphorylated.

Comparison of cytosolic and nuclear poly(A) polymerases from rat liver and a hepatoma: structural and immunological properties and response to NI-type protein kinases.

Poly(A) polymerases were purified from the cytosol fraction of rat liver and Morris hepatoma 3924A and compared to previously purified nuclear poly(A) polymerases. Chromatographic fractionation of the hepatoma cytosol on a DEAE-Sephadex column yielded approximately 5 times as much poly(A) polymerase as was obtained from fractionation of the liver cytosol. Hepatoma cytosol contained a single poly(A) polymerase species [48 kilodaltons (kDa)] which was indistinguishable from the hepatoma nuclear enzyme (48 kDa) on the basis of CNBr cleavage maps. Liver cytosol contained two poly(A) polymerase species (40 and 48 kDa). The CNBr cleavage patterns of these two enzymes were distinct from each other. However, the cleavage pattern of the 40-kDa enzyme was similar to that of the major liver nuclear poly(A) polymerase (36 kDa), and approximately three-fourths of the peptide fragments derived from the 48-kDa species were identical with those from the hepatoma enzymes (48 kDa). NI-type protein kinases from liver or hepatoma stimulated hepatoma nuclear and cytosolic poly(A) polymerases 4-6-fold. In contrast, the liver cytosolic 40- and 48-kDa poly(A) polymerases were stimulated only slightly or inhibited by similar units of the protein kinases. Antibodies produced in rabbits against purified hepatoma nuclear poly(A) polymerase reacted equally well with hepatoma nuclear and cytosolic enzyme but only 80% as well with the liver cytosolic 48-kDa poly(A) polymerase and not at all with liver cytosolic 40-kDa or nuclear 36-kDa enzymes. Anti-poly(A) polymerase antibodies present in the serum of a hepatoma-bearing rat reacted with hepatoma nuclear and cytosolic poly(A) polymerases to the same extent but only 40% as well with the liver cytosolic 48-kDa enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-RNA polymerase I antibodies: potential role in the induction and progression of murine lupus nephritis.

Antibodies against RNA polymerase I were detected in plasma and kidney eluates of NZB/W mice. Plasma concentrations of the antibodies were the highest in mice with incipient nephritis and the lowest in mice with progressive nephritis. Mice with attenuated nephritis due to immunosuppressive therapy had intermediate plasma concentrations of the antibodies. The specific concentrations (ng/microgram IgG) of anti-RNA polymerase I antibodies in kidney eluates were significantly (10- to 70-fold) greater than the corresponding plasma concentrations. These results indicated that the decreased plasma concentration of the antibodies in mice with more advanced disease was at least partially due to selective concentration of anti-RNA polymerase I antibodies in the kidneys. The degree of this selective concentration was directly proportional (R2 = 0.9962) to the severity of renal disease, as reflected by the concentration (microgram/g tissue) of IgG eluted from the kidneys. The concentration (microgram/g tissue) of anti-RNA polymerase I eluted from the kidneys also was increased in mice with more severe renal disease. Further, the extent of this increase was greater than that of total IgG, again suggesting that anti-RNA polymerase I antibodies had been selectively concentrated in the kidneys. These findings are strongly suggestive of an important role for the RNA polymerase I/anti-RNA polymerase I antibody system in the pathogenesis of murine lupus nephritis.

Structural and immunological identity of rat hepatoma and fetal liver nuclear poly(A) polymerase.

Poly(A) polymerase was partially purified from isolated nuclei of fetal rat liver. Antibodies produced in rabbits immunized with purified nuclear poly(A) polymerase from a rat hepatoma exhibited nearly identical affinity for the partially purified fetal liver and hepatoma enzymes. The extent of the antibody reaction with adult liver nuclear poly(A) polymerase partially purified in a similar manner was only 1.4% of that obtained with the hepatoma enzyme. Immune complex formation was observed between the antibodies and a major polypeptide in the fetal liver enzyme preparation which corresponded to the hepatoma enzyme (mol. wt. 48 000). No other polypeptide in the fetal liver enzyme preparation reacted with the antibodies. The 48-kDa fetal liver polypeptide produced a CNBr cleavage pattern identical to that of hepatoma poly(A) polymerase which is known to be different from the cleavage pattern of the adult liver major nuclear poly(A) polymerase. A fetal liver polypeptide corresponding to the adult liver enzyme (mol. wt. 38 000) was not evident. These results coupled with other data suggest that the hepatoma nuclear poly(A) polymerase is an oncofetal protein.

Immunization of rabbits with purified RNA polymerase I induces a distinct population of antibodies against nucleic acids as well as anti-RNA polymerase I antibodies, both characteristic of systemic lupus erythematosus.

Rabbits were immunized with either RNA polymerase I or poly(A) polymerase that had been purified to apparent homogeneity and was devoid of nucleic acids. Sera from rabbits thus immunized were screened for antibodies against nucleic acids. All seven rabbits injected with RNA polymerase I but none of the four rabbits immunized with poly(A) polymerase produced anti-nucleic acid antibodies. Anti-RNA polymerase I antibodies were induced after a single injection of the enzyme. Anti-polynucleotide antibodies were not detectable until after the second immunization. Anti-RNA polymerase I antibodies could be detected with as little as 100 pg of purified RNA polymerase I in the radioimmunoassay. At least 50 ng of poly(A) or 200 ng of DNA was required to detect anti-nucleic acid antibodies. The immunoreactivity of anti-RNA polymerase I antisera was greater with synthetic polynucleotides than with DNA, particularly early in the immunization schedule. Alkaline phosphatase treatment of poly(A) to remove 5' phosphates nearly abolished its antigenicity with respect to the early sera and decreased antibody binding of later sera by 60%. These results indicate that the anti-nucleic acid antibodies produced early were primarily directed against determinants including the 5'-terminal phosphates while antibodies produced later were directed against other sites. The antinucleic acid antibodies and anti-RNA polymerase I antibodies formed two distinct populations that were not immunologically crossreactive. We suggest that after injection, RNA polymerase I becomes associated with the nucleic acids present in blood plasma which renders them immunogenic; thus, association of nucleic acids with autoimmunogenic RNA polymerase I may be one of the mechanisms by which anti-DNA antibodies are induced in systemic lupus erythematosus.

Phosphorylation of deoxyribonucleic acid dependent RNA polymerase II by nuclear protein kinase NII: mechanism of enhanced ribonucleic acid synthesis.

RNA polymerase II was purified from Morris hepatoma 3924A by a series of ion-exchange and affinity column chromatographic fractionations, followed by sucrose gradient centrifugation in the presence of 0.3 M KC1. Purified RNA polymerase II had a specific activity of greater than 400 nmol of UMP incorporated (30 min)-1 (mg of protein)-1 by using double-stranded DNA as template. The purified enzyme contained five polypeptides (Mr 214 000, 140 000, 33 000, 25 000, and 21 000) that were present in molar quantities and two additional polypeptides (Mr 19 000 and 18 000) that had a combined molar ratio of 1.0. The cyclic AMP independent nuclear protein kinase NII, also purified from hepatoma 3924A, was able to phosphorylate RNA polymerase II polypeptides of Mr 214 000, 140 000, and 21 000. Phosphorylation of the polymerase was accompanied by enhanced transcription of double-stranded DNA, heat-denatured DNA, and poly[d-(A-T)]. The elevation in RNA polymerase activity was dependent upon the presence of hydrolyzable ATP and resulted from an increased number of RNA molecules synthesized in vitro. The average length of RNA chains was not affected by the kinase. Under similar conditions, protein kinase NII also stimulated homologous RNA polymerase I. In contrast to the phosphorylation of polymerase II, modification of polymerase I resulted in an increase in the average size, but not number, of RNA chains synthesized. The specificity of the NII kinase-catalyzed reaction was demonstrated by the inability of another homologous protein kinase, NI, to phosphorylate or activate RNA polymerase II.

Cysteine biosynthesis in a fungus, Histoplasma capsulatum.

We have analyzed a step in cysteine biosynthesis in several strains of the pathogenic dimorphic fungus, Histoplasma capsulatum. Mycelial cells of all strains tested are prototrophic. However, the yeast phase cells of most stains do not grow in the absence of -SH-containing compounds due to the apparent lack of an active form of sulfite reductase, a crucial enzyme in the cysteine biosynthetic pathway. In contrast, the yeast phase cells of one strain (Downs) have been found to have an active sulfite reductase and can grow in the absence of cysteine if serine is added. A different metabolic block must thus exist in this strain. Sulfite reductase in the yeast form of Downs strain is completely repressed by growth on cysteine while the mycelial form seems to be constitutive. The yeast and mycelial phase extracts were analyzed on polyacrylamide gels. A distinct protein band appeared in extracts prepared from the yeast cells incubated in minimal or serine-containing media, but not in extracts from mycelia or from cysteine-grown yeast cells.

Structurally and immunologically distinct poly(A) polymerases in rat liver. Occurrence of a tumor-type enzyme in normal liver.

Poly(A) polymerases purified from rat liver nuclei consisted of two distinct species, a predominant enzyme of Mr = 38,000 and a minor one of Mr = 48,000. Prior to extensive purification, the minor enzyme constituted approximately 1% of the total liver poly(A) polymerase. Poly(A) polymerase purified from a rat tumor, Morris hepatoma 3924A, was comprised of a single species of Mr = 48,000 which was identical to the minor liver enzyme with respect to chromatographic and immunological characteristics. Gel filtration on Sephacryl S-200 using 0.3 M NaCl for elution showed that the major liver poly(A) polymerase had a molecular weight of 156,000, which corresponded to a tetramer of the 38-kDa polypeptide, whereas the hepatoma and minor liver 48-kDa species existed as dimers with a molecular weight of 96,000. Fractionation by Sephacryl S-200 resulted in complete loss of both liver poly(A) polymerase activities which could be restored by exogenous N1-type protein kinase. Following CNBr cleavage, the 48-kDa poly(A) polymerase from liver and hepatoma exhibited nearly identical peptide maps which were distinct from that of the major liver enzyme (38 kDa). Antibodies raised against tumor poly(A) polymerase reacted with the 48-kDa polypeptide but not with the 38-kDa liver enzyme. Immune complex formation was observed between seven of the eight CNBr cleavage products derived from the 48-kDa polypeptide of both liver and hepatoma. It is concluded that distinct genes in rat liver code for two structurally and immunologically unique nuclear poly(A) polymerases, one of which is identical to the enzyme from the hepatoma.

Phosphorylation of RNA polymerase I augments its interaction with autoantibodies of systemic lupus erythematosus patients.

Purified RNA polymerase I was phosphorylated by the endogenous protein kinase or dephosphorylated by alkaline phosphatase and used as antigen in a radioimmunoassay with sera from systemic lupus erythematosus patients or serum from an immunized rabbit. Enzyme incubated in the absence of ATP or phosphatase served as control. Three to seven times more of the autoantibodies in the patients' sera reacted with phosphorylated RNA polymerase I than with control enzyme. The reactivity of the dephosphorylated enzyme with lupus autoantibodies was only 50-60% of that observed with control enzyme. Neither phosphorylation nor dephosphorylation of the enzyme had an effect on its reaction with the rabbit antibodies. The effect of phosphorylation on the reaction of each RNA polymerase I subunit (S1-S8; Mr = 190,000-17,000) with the patients' antibodies was determined by an immunoblot procedure following resolution of the subunits on polyacrylamide gels. Prior phosphorylation of the enzyme resulted in a dramatic increase in binding of each patient's antibodies to all polymerase subunits with the exception of S4. Anti-S4 antibody was not detected with either phosphorylated or control enzyme. Strikingly, antibodies in each patients' sera reacted with S6 only after its phosphorylation. Similarly, anti-S5 antibodies in the serum of one patient were only detected with phosphorylated RNA polymerase I. The present data suggest that at least a significant fraction of the anti-RNA polymerase I autoantibodies in the sera of systemic lupus erythematosus patients might be directed against phosphorylated sites on the enzyme and that phosphorylation may have a role in the production of this and other autoimmunogenic nuclear components which are hallmarks of this disease.