A 52-kD protein is a novel component of the SS-A/Ro antigenic particle.

Anti-SS-A/Ro autoantibodies are found in the sera of patients with Sjogren's syndrome (SS) and SLE. In the course of analyzing 61 SS patients for their autoantibody profiles, we found that 42 were positive for anti-SS-A by double diffusion in agarose and demonstrated precipitin lines identical to that produced by a prototype anti-SS-A serum. Further analysis of these SS-A antibody-positive sera by Western blotting of cell extracts revealed that 21 sera reacted with two proteins of 60 and 52 kD, 13 sera reacted with 52-kD protein, two detected only 60 kD, while six were nonreactive. Affinity-purified anti-60-kD and anti-52-kD antibodies reacted exclusively with their corresponding antigens. Partial proteolysis of these proteins did not reveal common degradation fragments. Thus the 52- and 60-kD proteins were found to be antigenically and apparently structurally distinct from each other. They were also distinct from 48-kD SS-B/La protein. In immunoprecipitation using labeled cell extracts, affinity-purified anti-52-kD antibodies brought down the 52-kD protein as well as the 60-kD band. In [32P]orthophosphate-labeled HeLa cell extract both antibodies precipitated the same spectrum of small RNAs (hYl-5). In indirect immunofluorescence, anti-52-kD and anti-60-kD antibodies immunolocalized in similar subcellular structures and showed similar punctate nuclear staining patterns. Western blot analysis revealed that both proteins were present in lymphocytic as well as epithelial human cell lines tested. The data above define a new antigen of 52 kD which is another component of the SS-A particle and is associated in complex formation with the previously reported 60-kD protein.

Identification of the complement decay-accelerating factor (DAF) on epithelium and glandular cells and in body fluids.

Decay-accelerating factor (DAF) is a 70 kD membrane regulatory protein that prevents the activation of autologous complement on cell surfaces. Using immunohistochemical methods and a radioimmunometric assay based on mAbs to DAF, we found large amounts of membrane-associated DAF antigen on the epithelial surface of cornea, conjunctiva, oral and gastrointestinal mucosa, exocrine glands, renal tubules, ureter and bladder, cervical and uterine mucosa, and pleural, pericardial and synovial serosa. Additionally, we detected soluble DAF antigen in plasma, tears, saliva, and urine, as well as in synovial and cerebrospinal fluids. While plasma, tear, and saliva DAF are larger than erythrocyte (Ehu) membrane DAF by Western blot analysis, urine DAF is slightly smaller (67,000) in Mr. Unlike purified Ehu DAF, however, urine DAF is unable to incorporate into the membrane of red cells. Although its inhibitory activity on the complement enzyme C3-convertase is lower than that of Ehu DAF, it is comparable to that of serum C4 binding protein (C4bp). Biosynthetic studies using cultured foreskin epithelium and Hela cells disclosed DAF levels (approximately 2 X 10(5) molecules/cell) exceeding those on blood cells. In addition, these studies revealed the synthesis of two DAF species, one with apparent Mr corresponding to that of epithelial cell membrane DAF and the other to urine DAF, suggesting that the urine DAF variant arises from adjacent epithelium. The function of DAF in body fluids is unknown, but the observation that urine DAF has C4bp-(or factor H-)like activity shows that it could inhibit the fluid phase activation of the cascade.

Pathogenesis of shigella diarrhea. XI. Isolation of a shigella toxin-binding glycolipid from rabbit jejunum and HeLa cells and its identification as globotriaosylceramide.

A glycolipid that specifically binds shigella toxin was isolated from both HeLa cells and rabbit jejunal mucosa and identified as globotriaosylceramide (Gb3) by its identical mobility on HPTLC to authentic erythrocyte Gb3. Toxin also bound to a band tentatively identified as alpha-hydroxylated Gb3. In addition, toxin bound to P1 antigen present in group B human erythrocyte glycolipid extracts. The common feature of the three binding glycolipids is a terminal Gal alpha 1----4Gal disaccharide linked beta 1----4 to either Glc or GlcNAc. Globoisotriaosylceramide, which differs from Gb3 only in possessing a Gal alpha 1----3Gal terminal disaccharide, and LacCer, which lacks the terminal Gal residue of Gb3, were incapable of binding the toxin. Binding was shown to be mediated by the B subunit by the use of isolated toxin A and B subunits and monoclonal subunit-specific antibodies. Gb3-containing liposomes competitively inhibited the binding of toxin to HeLa cell monolayers but did not inhibit toxin-induced cytotoxicity. These studies show an identical carbohydrate-specific glycolipid receptor for shigella toxin in gut and in HeLa cells. The toxin B subunit that mediates this binding has also been shown to recognize a glycoprotein receptor with different sugar specificity. Thus, we have demonstrated that the same small (Mr 6,500) B subunit polypeptide has two distinctive carbohydrate-specific binding sites. The Gal alpha 1----4Gal disaccharide of the glycolipid toxin receptor is also recognized by the Gal-Gal pilus of uropathogenic E. coli. This suggests the possibility that the pilus and toxin B subunit contain homologous sequences. If this is true, it may be possible to use the purified Gal-Gal pilus to produce toxin-neutralizing antibodies.

Microtubule-associated proteins of HeLa cells: heat stability of the 200,000 mol wt HeLa MAPs and detection of the presence of MAP-2 in HeLa cell extracts and cycled microtubules.

One of the major groups of microtubule-associated proteins (MAPs) found associated with the microtubules isolated from HeLa cells has a molecular weight of just over 200,000. Previous work has demonstrated that these heLa MAPs are similar in several properties to MAP-2, one of the major MAPs of mammalian neural microtubules, although the two types of proteins are immunologically distinct. The 200,000 mol wt HeLa MAPs have now been found to remain soluble after incubation in a boiling water bath and to retain the ability to promote tubulin polymerization after this treatment, two unusual properties also shown by neural MAP-2. This property of heat stability has allowed the development of a simplified procedure for purification of the 200,000 HeLa MAPs and has provided a means for detection of these proteins, even in crude cell extracts. These studies have also led to the detection of a protein in crude extracts of HeLa cells and in cycled HeLa microtubules which has been identified as MAP-2 on the basis of (a) comigration with calf brain MAP-2 on SDS PAGE, (b) presence in purified microtubules, (c) heat stability, and (d) reaction with two types of antibodies prepared against neural high molecular weight-MAPs, one of these a monoclonal antibody against hog brain MAP-2, although present in HeLa cells, is at all stages of microtubule purification a relatively minor component in comparison to the 200,000 HeLa MAP's.

Effect of temperature of aldehyde fixation on the radioautographic localization of ribonucleoprotein in nucleoli of HeLa cells. Inhibition by puromycin and actinomycin D.

In efforts to clarify the role of the nucleolus and substructures thereof in the assembly or synthesis of protein associated with formation of the complete ribosome, the effect of variation of some conditions of aldehyde fixation on the intranuclear distribution of lysine-(3)H, arginine-(3)H, and uridine-(3)H was studied by differential grain count in radioautographs of PPLO-free HeLa cells. It was found that the nucleolus is a site of rapid assembly or synthesis of a protein, the synthesis of which is inhibited equally by puromycin (200 microg/ml) and by actinomycin D under conditions inhibitory for ribosomal precursor RNA synthesis (P < 0.01). This protein is fixed by phosphate-buffered formalin or glutaraldehyde at pH 7.3, but the label is diminished by fixation in customarily employed acetic ethanol or in formalin at acid pH. Elevation of temperature of formalin or glutaraldehyde fixatives to 37 degrees C consistently reduces the nucleolar protein label, but not the RNA label, by a proportion identical with that incurred by puromycin or actinomycin inhibition. This proportional reduction of nucleolar protein label occurs without evident loss of total grain count and is independent of length of fixation between 30 min and 4 hr, but it is not observed at 23 degrees C. The data support the interpretation that the proportion of nucleolar protein not fixed at 37 degrees C is associated with nucleolar ribosomal RNA but that it is dissociated at 37 degrees C in formalin or glutaraldehyde fixatives, probably on the basis of ionic dissociation of a conjugated ribonucleoprotein.

Distribution of newly formed ribosomal proteins in HeLa cell fractions.

The distribution of newly formed ribosomal proteins between cytoplasmic, nucleoplasmic, and nucleolar fractions of HeLa cells was determined. All but a few of the newly formed ribosomal proteins were concentrated 10- to 50-fold in the nucleolus and two- to fivefold in the nucleoplasm. Nevertheless, substantial amounts were found in the cytoplasm. Pretreatment of cells with actinomycin D to deplete the nucleolar pool of ribosomal precursor RNA had no effect on the concentration of newly formed ribosomal proteins in the nucleus, but did lead to an increased amount in the nucleoplasm at the expense of the nucleolus.

Large species differences in the pattern of snPI RNA which can distinguish ape from human.

The snPI RNA species are a recently described set of molecules whose sizes range from 5S to 10S. They can be labeled in vitro in isolated nuclei and are apparently formed by an RNA polymerase I type of activity. However, in contrast to ribosomal precursor RNA, the usual polymerase I product, they are not found in the nucleolus but rather are located in the nucleoplasm. The snPI RNAs have been found in all mammalian cell types studied. The spectrum seen in gel electrophoresis is unique to each animal species studied but is essentially the same in different cell types within a species. The differences in snPI patterns are quite large between even closely related species and are clearly distinguishable in gorilla and human cells.

Mapping nucleolar proteins with monoclonal antibodies.

Using monoclonal antibodies as probes, we have characterized three antigens with respect to localization in the nucleolus, molecular weight and solubility. Two proteins, of 110,000 and 94,000 apparent molecular weight, were found associated with the ribonucleoprotein fibers. A third protein, with a molecular weight of 40,000, was accumulated at the nucleolar periphery, was present in the nucleoplasm, and may be involved in pre-ribosome maturation and transport.

Monoclonal antibody characterization of the C proteins of heterogeneous nuclear ribonucleoprotein complexes in vertebrate cells.

The C proteins (C1 and C2) are major constituents of the 40S subparticle of heterogeneous nuclear ribonucleoprotein complexes (hnRNPs) (Beyer, A.L., M.E. Christensen, B.W. Walker, and W.M. LeStourgeon, 1977, Cell, 11:127-138) and are two of the most prominent proteins that become cross-linked by ultraviolet light to heterogeneous nuclear RNA (hnRNA) in vivo. Studies are described here on the characterization of the C proteins in vertebrate cells using monoclonal and polyclonal antibodies. Monoclonal antibodies to genuine RNP proteins, including the C proteins, were obtained by immunizing mice with purified complexes of poly(A)+ hnRNA and poly(A)+ mRNA with their contacting proteins in vivo obtained by ultraviolet cross-linking the complexes in intact cells (Dreyfuss, G., Y.D. Choi, and S.A. Adam, 1984, Mol. Cell. Biol., 4:1104-1114). One of the monoclonal antibodies identified the C proteins in widely divergent species ranging from human to lizard. In all species examined, there were two C proteins in the molecular weight range of from 39,000 to 42,000 for C1, and from 40,000 to 45,000 for C2. The two C proteins were found to be highly related to each other; they were recognized by the same monoclonal antibodies and antibodies raised against purified C1 reacted also with C2. In avian, rodent, and human cells the C proteins were phosphorylated and were in contact with hnRNA in vivo. Immunofluorescence microscopy demonstrated that the C proteins are segregated to the nucleus. Within the nucleus the C proteins were not found in nucleoli and were not associated with chromatin as seen in cells in prophase. These findings demonstrate that C proteins with similar characteristics to those in humans are ubiquitous components of hnRNPs in vertebrates.

U1 small nuclear ribonucleoprotein studied by in vitro assembly.

The small nuclear RNAs are known to be complexed with proteins in the cell (snRNP). To learn more about these proteins, we developed an in vitro system for studying their interactions with individual small nuclear RNA species. Translation of HeLa cell poly(A)+ mRNA in an exogenous message-dependent reticulocyte lysate results in the synthesis of snRNP proteins. Addition of human small nuclear RNA U1 to the translation products leads to the formation of a U1 RNA-protein complex that is recognized by a human autoimmune antibody specific for U1 snRNP. This antibody does not react with free U1 RNA. Moreover, addition of a 10- to 20-fold molar excess of transfer RNA instead of U1 RNA does not lead to the formation of an antibody-recognized RNP. The proteins forming the specific complex with U1 RNA correspond to the A, B1, and B2 species (32,000, 27,000, and 26,000 mol wt, respectively) observed in previous studies with U1 snRNP obtained by antibody-precipitation of nuclear extracts. The availability of this in vitro system now permits, for the first time, direct analysis of snRNA-protein binding interactions and, in addition, provides useful information on the mRNAs for snRNP proteins.

Actin associated with membranes from 3T3 mouse fibroblast and HeLa cells.

A protein component of membranes isolated from 3T3 mouse fibroblasts and HeLa cells has been identified as actin by peptide mapping. Extensive but apparently not total coincidence was found between the peptide maps of these two nonmuscle membrane-associated actins compared to chick skeletal muscle actin. Between 2 and 4 percent of the total membrane protein appears in the actin band on sodium dodecyl sulfate polyacrylamide gels of 3T3 membranes while about 4 percent of the membrane protein appears as the actin band from HeLa membranes. These values represent approximately the same proportion of actin to total protein found in the cell homogenates. Treatment of intact cells with levels of cytochalasin B sufficient to cause pronounced morphological changes did not change the amount of actin associated with the membrane in either 3T3 or HeLa cells. However, incubation of isolated membranes under conditions favoring conversion of actin from filamentous to monomeric form resulted in dissociation of approximately 80 and 60 percent of the actin from 3T3 and HeLa membranes, respectively. Thus, approximately 20 percent of 3T3 membrane actin and 40 percent of HeLa membrane actin remained associated with the membrane even under actin depolymerizing conditions.

HeLa cell plasma membranes. I. 5'-Nucleotidase and ouabain-sensitive ATPase as markers for plasma membranes.

A method for the preparation of HeLa cell plasma membrane ghosts is described. The purity of the plasma membrane fraction was examined by phase contrast and electron microscopy, by chemical analysis, and by assay of marker enzymes. Data on the composition of the plasma membrane fraction are given. It was observed that the distribution pattern of 5'-nucleotidase activity among the subcellular fractions differed from that of ouabain-sensitive ATPase. In addition, the specific activity of 5'-nucleotidase did not follow the distribution of the membrane ghosts. Thus, this enzyme would seem unsuitable as a plasma membrane marker. A complete balance sheet for marker enzyme activities during the fractionation is necessary for the calculation of increase in specific activity because the activities of both 5'-nucleotidase and ouabain-sensitive ATPase might change during the fractionation procedures.

Fos-associated protein p39 is the product of the jun proto-oncogene.

The Fos protein complex and several Fos-related antigens (FRA) bind specifically to a sequence element referred to as the HeLa cell activator protein 1 (AP-1) binding site. A combination of structural and immunological comparisons has identified the Fos-associated protein (p39) as the protein product of the jun proto-oncogene (c-Jun). The p39/Jun protein is one of the major polypeptides identified in AP-1 oligonucleotide affinity chromatography extracts of cellular proteins. These preparations of AP-1 also contain Fos and several FRA's. Some of these proteins bind to the AP-1 site directly whereas others, like Fos, appear to bind indirectly via protein-protein interactions. Cell-surface stimulation results in an increase in c-fos and c-jun products. Thus, the products of two protooncogenes (and several related proteins), induced by extracellular stimuli, form a complex that associates with transcriptional control elements containing AP-1 sites, thereby potentially mediating the long-term responses to signals that regulate growth control and development.

5-methylcytidylic acid: absence from mitochondrial DNA of frogs and HeLa cells.

The content of 5-methylcytidylic acid in nuclear DNA and mito chondrial DNA of Xenopus laevis and HeLa cells has been determined. Both nuclear DNA's contain 5-methylcytidylic acid. The 5-methylcytidylic acid content of X. laevis DNA is 1.7 mole percent of total nucleotides, and that of HeLc cell DNA is 0.7 mole percent. In neither mitochondrial DNA could any 5-methylcytidylic acid be detected; the limit of sensitivity was judged at below 0.1 mole percent for X. laevis DNA and below 0.05 mole percent for HeLa cell DNA.

Polyadenylic acid sequences in the virion RNA of poliovirus and Eastern Equine Encephalitis virus.

Poliovirus virion RNA contains a single covalently bound sequence of polyadenylic acid which is approximately 49 nucleotides long. A single, slightly longer polyadenylic acid sequence is contained in Eastern Equine Encephalitis virus RNA. Since these viruses are otherwise dissimilar these sequences may play a common role in viral replication, possibly in translation of the viral RNA.

Human Ro ribonucleoprotein particles: characterization of native structure and stable association with the La polypeptide.

Anti-Ro autoantibodies, found in sera of patients with systemic lupus erythematosus, Sjogren's syndrome, and related diseases, target the Ro ribonucleoprotein particles (RNPs). Although the polypeptide and RNA components of the Ro RNPs have been characterized, much less is known about the native structure of these particles. We have now characterized by biochemical techniques intact Ro ribonucleoprotein particles from cultured HeLa cells. These particles segregated in three discrete subpopulations with characteristic physicochemical properties: one containing hY5 RNA (RohY5 particles), one containing only hY4 RNA (RohY4 particles) and one with hY1, hY3, and hY4 RNAs (RohY1-hY4 particles). The RohY5 particles were purified free of contaminating ribonucleoproteins; both the La and the 60-kD Ro polypeptides were stable components of this portion of the Ro RNPs. The La RNPs co-purified with the RohY4 particles and contaminated the RohY1-hY4 RNPs. The stable association between the La and the 60-kD Ro polypeptides provides a potential macromolecular target for the linked set of anti-Ro and anti-La antibodies, and suggests a possible functional association of these polypeptides.

Antibodies from patients with connective tissue diseases bind specific subsets of cellular RNA-protein particles.

We characterized the RNA-containing antigens precipitated by sera from 260 patients with positive antinuclear antibodies. 49 individuals, most of whom had systemic lupus erythematosus or Sjögren's syndrome, possessed antibodies that precipitated the previously identified RNP, Sm, Ro, and La antigens either singly or in combinations. These antigens, which are located on discrete sets of small nuclear or cytoplasmic RNA-protein particles, exhibited a number of antigenic interrelationships. One patient's serum recognized a new particle containing a small RNA which we have called Th; it also precipitated the Ro complexes. Other patients with systemic lupus erythematosus, hepatitis B virus infection, juvenile rheumatoid arthritis, myositis, and rheumatoid arthritis had antibodies that precipitated specific subsets of ribosomal RNA and transfer RNA. One patient's serum contained a monoclonal immunoglobulin G that precipitated ribosomes. Most of these antibodies identified antigenic determinants constituted at least in part of protein. The specificity of the proteins bound to particular cellular RNA, probably explains the exquisite precision with which antibodies from rheumatic disease patients discriminate among RNA subsets. Such sera should be useful probes for investigating specific roles that different RNA and RNA-protein complexes play in cellular metabolism.

Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities.

Several proteins of heterogeneous nuclear ribonucleoprotein (hnRNP) particles display very high binding affinities for different ribonucleotide homopolymers. The specificity of some of these proteins at high salt concentrations and in the presence of heparin allows for their rapid one-step purification from HeLa nucleoplasm. We show that the hnRNP C proteins are poly(U)-binding proteins and compare their specificity to that of the previously described cytoplasmic poly(A)-binding protein. These findings provide a useful tool for the classification and purification of hnRNP proteins from various tissues and organisms and indicate that different hnRNP proteins have different RNA-binding specificities.

Quantitation and intracellular localization of the 85K heat shock protein by using monoclonal and polyclonal antibodies.

Two monoclonal antibodies have been produced against the human 85,000-molecular-weight heat shock protein (hsp85). One of these, 16F1, cross-reacts with the murine homolog and is shown by peptide map immunoblots to be directed against an epitope different from that recognized by the other monoclonal antibody, 9D2. Both monoclonal antibodies recognize only a single Mr-85,000 species in two-dimensional immunoblots. Immunoprecipitation did not reveal an association of this heat shock protein with any other protein in HeLa cells. Immunoperoxidase staining showed a purely cytosolic distribution at both light and electron microscopic levels and no association with membranes, mitochondria, or other organelles. The 9D2 monoclonal and a polyclonal antimurine hsp85 antibody were used to identify the antigens and to quantitate their levels in a variety of normal tissues by immunoautoradiography. Relative abundance in the various tissues as determined by Coomassie blue staining correlates reasonably well with the immunoreactivity. Testis and brain, for example, have high hsp85 levels, whereas heart and skeletal muscle have little or none. The Mr-85,000 sodium dodecyl sulfate-polyacrylamide gel band in testis and brain lysates was further confirmed to be hsp85 by one-dimensional partial proteolytic peptide mapping. Based on these data and our previous observations showing that synthesis and levels of the protein are altered by depriving cultured cells of glucose, we speculate that intracellular hsp85 levels depend on differences in the intermediary metabolism of glucose in the various tissues. Furthermore, it appears that high basal levels of this heat shock protein may not necessarily protect cells against heat shock, since testis is one of the most heat-sensitive tissues and has the highest hsp85 level.

High-resolution mapping of S1- and DNase I-hypersensitive sites in chromatin.

A new and easy technique for accurately mapping DNase I- and S1 nuclease-hypersensitive sites is described. The technique is a modification of primer extension and S1 nuclease methods conventionally used to map RNA ends.