Novel DNA-protein complex and a large DNA in SLE cryoprecipitates.

Agarose gel electrophoresis of cryoprecipitates from systemic lupus erythematosus (SLE) patients revealed the presence of a slowly migrating DNAse I-sensitive DNA species at the top of the gel. Upon deproteinization, electrophoretic migration was modified favouring the migration of a 17.5 kb DNA fragment. Mixing experiments adding human serum or plasma to a lambda phage DNA digest revealed a DNA-protein interaction shown by an accumulation of high mol. wt polynucleotide at the top of the gels, and a slowed migration of the DNA bands. No comparable effect was observed when serum albumin was added to the lambda DNA digest. Dot hybridization analysis showed preferential reactivity of the 17.5 kb DNA to human DNA, implying its human origin. Our data suggests that most of this high molecular weight DNA exists as a DNA-2 protein complex. Our mixing experiments also suggest the occurrence of an excess of free DNA antibodies. We propose that the DNA-protein association may play a role in the stabilization and immunogenicity of the nucleoprotein complex.     

An adenovirus protein associated with the ends of replicating DNA molecules.

The adenovirus DNA-protein complex contains the linear DNA molecule and a 55K protein covalently attached to each 5′ end. We describe a simple assay for adenovirus DNA-protein complex, in which DNA, covalently linked to the terminal protein, specifically binds to benzoylated naphthoylated DEAE-cellulose. The DNA-protein complex can be recovered intact from the column by elution with urea and SDS, or the DNA moiety can be eluted after incubation of the column with protease. Using this assay system, we have shown that protein is associated with pulse-labeled single strands of DNA from the terminal restriction enzyme fragments of replicating adenovirus DNA. We suggest that the terminal protein has a function in DNA replication.     

The infectivity of adenovirus 5 DNA-protein complex.

Adenovirus 5 DNA-protein complexes were prepared by treating virions with 4 M guanidinium chloride and resolving the faster sedimenting viral DNA from released capsid protein. These complexes were characterized by both gel electrophoresis and electron microscopy. After dialysis into saline buffer, approximately one-half of the viral DNA-protein complex was aggregated by association at its termini into conformations other than linear monomer length duplex molecules. However, the monomer length linear molecules in the viral DNA-protein complex sample also have protein attached to both of their termini: These linear molecules are resistant to digestion by a pressessive nuclease, adenosine triphosphate-dependent deoxyribonuclease, that requires a free terminus for activity, while Pronase-treated adenovirus DNA is susceptible to digestion, and the restriction endonuclease cleavage fragments from both ends of the viral DNA-protein complex migrate at an anomalous rate during electrophoresis in an agarose gel. As the infectivity of Ad5 DNA is exceptionally low, the infectivity of the DNA-protein complex was tested using the calcium technique for transfection. The efficiency of transfection with the Ad5 DNA-protein complex is about 100-fold higher than that of Pronase-treated Ad5 DNA.     

Human Membrane-bound C3 Receptors

The aim of the present study was to define the physicochemical structure of C3b and C3d receptors of lymphoid cells, C3b and C3b receptors were isolated from KBr lysates of the 20,000 g fraction of human tonsil homogenates by immunoprecipitation with an anti-C3 receptor serum (AC3RS). Sodium dodecyl sulphate (SDS) gel filtration and polyacrylamide gel electrophoresis (PAGE) of unreduced immunoprecipitates revealed a highly predominant component with an apparent molecular weight (mol.wt.) greater than 1 × 10⁴ and a small component with a mol.wt. of 80,000. After reduction, the SDS-PAGE profile was made up of a constant major 38,000 mol.wt. component and an inconstant smaller 18,000 mol.wt. component. The 38,000 (and also the 18,000) component could he isolated only from C3 receptor-active lysates, and not from C3 receptor-negative lysates. Taken together, the results of this study suggest that the active C3 receptor molecule of tonsil cells is a lipoprotein complex with a mol.wt. greater than 1 × 10⁴; its protein moiety consists predominantly of disulphide-bridged polypeptide chains with a mol.wt, of 38,000; C3b and C3d receptors are composed of equal-sized polypeptide chains, but the specific binding sites for C3b and C3d are located on different molecules.     

Poliovirus polypeptides examined in more detail.

The pattern of viral polypeptide synthesis and cleavage in poliovirus-infected cells was shown by autoradiography to be considerably more complex than previously thought. In normal growth, at least 26 distinct polypeptides were found, and various modifications of the cleavage process revealed a total of at least 34. Most of the new polypeptides were minor components that were unstable during a chase. Different cultural modifications led to different polypeptide ratios, and it appeared likely that several cleavage activities were involved . Minor differences were found in the polypeptide contents of cytoplasmic extracts and whole infected cells. The complexity of the cleavage pattern necessitated a new nomenclature based on mol. wt. (e.g. Pp110, "poliovirus protein" of 110000). Particular attention was paid to mol. wt. determinations, notably in the use of internal protein standards and more fully denaturing gel conditions. The size of the "primary translation product" of poliovirus RNA was found to be 210000 daltons, so that either 20% of the viral genome is not translated in vivo, or some is read as a smaller independent translation unit.     

The visualization of a circular DNA-protein complex from adenovirions

A DNA-protein complex was extracted from adenovirus type 5 virions with 4 M guanidine hydrochloride, and was purified by sucrose gradient velocity centrifugation. The isolated DNA-protein complex contained about 0.8% of the total protein of intact virions. Electron microscopic visualization of the DNA-protein complexes revealed circular DNA containing one protein entity as well as linear molecules with protein knobs at both molecular ends. The location of the protein knobs makes it probable that these knobs indicate the presence of a terminal protein discovered by 4., 5., which is able to keep linear adenovirus DNA molecules in a circular state.     

A model for vesicular exanthema virus, the prototype of the calicivirus group

The structure of vesicular exanthema virus, the prototype member of the calicivirus group, has been studied in more detail. The RNA comprises 18% of mol. wt. of about 2.8 x 10(6), based on polyacrylamide gel electrophoresis experiments in the presence of formaldehyde. The virus contains one major polypeptide, mol. wt. 70 x 10(3) as determined by polyacrylamide gel electrophoresis and by chromatography on Sepharose 6B in the presence of 6 M-guanidine. Further evidence for the presence of a single major polypeptide was obtained by tryptic peptide analysis of 35S-methionine labelled virus. The mol. wt. of a protein oligomer produced by adjusting the pH of virus suspensions to 3.5 was c. 200 x 10(3). On the basis of these data we propose a T = 3 model for the virus capsid incorporating 180 copies of the virus protein.     

Distinct regional distribution in the brain of messenger RNAs for the two isoforms of synaphin associated with the docking/fusion complex

Synaphin is a 19,000 mol. wt cytosolic protein we first found to co-purify with the docking/fusion complex crucial to neurotransmitter release from presynaptic terminals. Two isoforms of synaphin (synaphins 1 and 2) (also called complexins II and I, respectively) exist in the rat brain. On density gradient centrifugation of a Triton X-100 extract of brain membranes, synaphin was found to be associated with the 7S complex that contains synaptotagmin, syntaxin, synaptosomal-associated protein of 25,000 mol. wt and vesicle-associated membrane protein. A smaller complex devoid of synaphins was also identified by immunoprecipitation with a monoclonal antibody against synaptosomal-associated protein of 25,000 mol. wt. Messenger RNAs for synaphins 1 and 2 were expressed predominantly in the brain. In situ hybridization using probes specific to synaphins 1 and 2 indicated that the distribution of their mRNAs was significantly different in brain regions such as olfactory bulb, hippocampus, cerebral cortex, piriform cortex, cerebellum, thalamus and facial nuclei. These results show synaphin as a component of the 7S complex and suggest different physiological implications for the two isoforms.     

Virus-specific basic phosphoproteins associated with herpes simplex virus type a (HSV-1) particles and the chromatin of HSV-1-infected cells

Herpes simplex type 1 Angelotti (HSV-1 ANG) virions were shown to contain two major acid-soluble proteins, BP1 and 2, which by size and charge analysis were also found to be associated with chromatin isolated from HSV-1 ANG-infected African green monkey kidney cells (HSV-chromatin). BP1 and 2 proved to exist in a phosphorylated state both in virions and in HSV-chromatin. BP1 consisted of a single polypeptide of 38 K mol. wt. which was correlated to the tegument protein VP22. In SDS-polyacrylamide gels BP2 migrated as a single polypeptide band with an apparent mol. wt. of 12 K. Urea gel analysis revealed that BP2 consisted of three components, BP2a, b and c, of different phosphate contents. Arguments were provided that these components probably represent different polypeptides of similar mol. wt. HSV-chromatin, in addition to BP1, BP2a, b and c contained a further major virus-induced basic phosphopolypeptide of mol. wt. 65 K which was not detected in acid-extracts of mature virions.     

Physicochemical characterization of C3b receptors isolated from human erythrocytes by immunoprecipitation.

A high yield of active C3b receptors was obtained by solubilizing human erythrocyte membranes with 2 M KBr, whereas other solubilization agents yielded no, or significantly less activity. Gel filtration of the KBr lysates revealed that the apparent molecular wieght of biologically active C3b receptor molecules was greater than 1 x 10(6). Immunoprecipitates prepared with radio-iodinated KBr lysates and anti-C3 receptor sera (AC3RS) were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) or sodium dodecyl gel filtration. Unreduced SDS-PAGE and gel filtration profiles showed three predominant peaks with apparent mol. wts of 1--1.3 x 10(6), 80,000 and 60,000. Whereas the high mol. wt component decreased only slightly after reduction, the 80,000 and 60,000 mol. wt components disappeared and two new peaks with apparent mol. wts of 38,000 and 18,000 appeared in SDS-PAGE profiles. Although the high mol. wt component present in reduced SDS-PAGE profiles was detectable in some of the control experiments, none of the other peaks could be precipitated with control sera, and these components could be demonstrated only when KBr lysates of C3b receptor-positive erythrocytes and AC3RS that were able to inhibit ligand binding of the C3b receptors were used for precipitation. These findings suggest that (a) the C3b receptor of human erythrocytes in its biologically active state is a macromolecule with an apparent mol. wt higher than 1 x 10(6) and (b) the protein moiety consists predominantly of non-covalently linked protein molecules with apparent mol wts of 80,000 and 60,000. These protein molecules are composed of disulphide-bridged polypeptide chains with apparent mol. wts of 38,000 and 18,000.     

Binding of C-reactive protein to Aspergillus fumigatus fractions

Calcium-dependent binding of C-reactive protein (CRP) to Aspergillus fumigatus was determined by enzyme-linked immunosorbent assay. A homogenate of young hyphae was fractionated by hydrophobic interaction chromatography followed by gel filtration. High CRP-binding activity was found in a fraction of mol. wt c. 500,000 which was characterised by strong binding to the hydrophobic column. Three fractions of less conspicuous CRP-binding activity were identified (c. 500 000, 150 000 and 150 000-50 000 mol. wt respectively). In these four fractions, phosphorylcholine was detected by an anti-phosphorylcholine mouse hybridoma antibody. Some CRP-binding activity in fractions with low affinity for the hydrophobic column did not correspond closely with the presence of phosphorylcholine. It is suggested that C-reactive substance in A. fumigatus is heterogeneous. The C-reactive substances did not correspond with fractions containing major antigens (470 000 and 250 000 mol. wt respectively) which elicit a strong immune response in man.     

Isolation and Characterization of TR-c, an Antigen of the Reiter Treponeme Precipitating with Antibodies in Syphilis

TR-c is a Reiter treponemal antigen that cross-reacts with an antigen in Treponema pallidum (Nichols pathogenic strain). Sera from patients with secondary syphilis contain precipitating antibodies against TR-c. The isolation of TR-c from a crude bacterial sonicate involves five fractionation steps: anion exchange chromatography (DE-52 Whatman), gel filtration (Ac-A-22. Ultrogel), and affinity chromatography respectively on phenyl-Sepharose CL 4B, iminodiacetic acid-Sepharose CL 4B. and lysine-Sepharose 4B. The purified TR-c was enriched 320 times compared with the starting material. and the recovery was 22%, TR-c was shown to be a protein, it did not bind to a series of lectins. and by gel filtration and polyacrylamide gel electrophoresis (PAGE) the mol, wt was determined to be in the range of 630, 000–730.000. It was found by SDS-PAGE to be composed of identical subunits, each having a mol. wt of 48, 000. The isolated TR-c was immunochemically pure when tested in crossed immunoelectrophoresis against polyspecific anti-Reiter Ig. The purified TR-c antigen was used for production of a monospecific rabbit antiserum. Monospecific rabbit anti-TR-c gave strong fluorescence with both the Reiter treponeme and T.pallidum .     

Antibodies against distinct nuclear matrix proteins are characteristic for mixed connective tissue disease.

Specific nuclear proteins, separated according to their molecular weight (mol. wt) by polyacrylamide gel electrophoresis (PAGE) and subsequently transferred to nitrocellulose sheets, are able to bind antibodies in sera from patients suffering from different types of connective tissue diseases. Antibodies against a characteristic set of nuclear protein antigens are found in sera from patients with mixed connective tissue disease (MCTD). Screening of 21 MCTD sera revealed a typical immunoblot pattern with major protein antigens of mol. wt 70,000 (20/21) (not identical with the Scl-70 antigen characteristic for scleroderma), mol. wt 31,000 (17/21), two proteins around mol. wt 23,000 (15/21) and two around mol. wt 19,000 (10/21). The 70,000, 23,000 and 19,000 antigens appeared to be rather insoluble nuclear proteins (i.e. components of the nuclear matrix). On behalf of their structural character they were present in nuclei from several types of cells but only in low amounts detectable in salt extracts of thymus acetone powder. The presence of antibodies directed against the mol. wt 70,000 antigen correlated strongly with the diagnosis of MCTD. This 70,000 antigen is not identical with the RNP antigen, a soluble ribonuclease sensitive ribonucleoprotein, since antibodies against nuclear RNP can be separated from anti-nuclear matrix antibodies by affinity chromatography using immobilized thymus salt extract. The distinct character of soluble nuclear RNP and structural nuclear matrix antigens is further supported by the fact that from 14 other anti-RNP sera obtained from patients with systemic lupus erythematosus (SLE), only three contained antibodies against the mol. wt 70,000 protein. Since the immunoblot pattern obtained with MCTD sera mostly was clearly distinguishable from the patterns obtained with sera from patients with related connective tissue diseases our results suggest that the immunoblotting technique might be useful as a diagnostic tool and support the concept of MCTD as a distinct entity.     

Characterization of a non-functional form of C1q found in patients with a genetically linked deficiency of C1q activity

A genetically defective form of C1q was purified from the sera of patients suffering from an immune complex related disease and who were homozygous for the defect. The defective C1q was haemolytically inactive and did not bind to immune aggregates or IgG-Sepharose. It showed the following similarities to the normal C1q molecule: a high glycine content and the presence of hydroxyproline and hydroxylysine; subunits with apparent mol. wts of 70,000 and 56,000, when examined by SDS-polyacrylamide gel electrophoresis under non-reducing conditions; preferential incorporation of 125I-label into only one of the types of chain present in the molecule, in a manner similar to that found for the C-chain of normal C1q. However, the defective molecule had an apparent mol. wt of approximately 155,000 in non-dissociating conditions, which is approximately one-third of the mol. wt of the normal molecule. Also, the material in the defective molecule preparation which corresponded, on the basis of mol. wt, to the disulphide-linked A-chain-B-chain dimer of normal C1q differed from that found in the normal molecule in that it did not appear to be sensitive to reducing agents. Collagenase and pepsin treatment of specific immunoprecipitates containing the radiolabelled defective molecule indicated that it is, like the normal molecule, composed of collagenous and non-collagenous domains.     

Pneumococcal bacteriophage Cp-1 contains a protein bound to the 5′ termini of its DNA

The genome of the pneumococcal bacteriophage Cp-1 has been isolated as a DNA-protein complex. The transfecting activity of this complex is destroyed by treatment with proteolytic enzymes. The DNA-protein complexes do not enter into agarose or acrylamide gels and are retained on glass fiber filters. The protein is specifically associated with the two 5′ termini of Cp-1 DNA on the basis of experiments carried out with restriction endonucleases, exonucleases, and radioactive labeling with [γ-³²P]ATP and polynucleotide kinase. The protein component, iodinated in vitro with ¹²⁵I, has a molecular weight of 28,000 determined by SDS-polyacrylamide gel electrophoresis. The protein remains associated with the Cp-1 DNA after thermal or alkali denaturation, incubation with 6 M guanidinium chloride or 8 M urea, and boiling in 2% SDS, 2% mercaptoethanol, and 6 M urea. When the complex was incubated in 1 M sodium hydroxide or 2.5 M piperidine only a partial breakage of the DNA-protein bond was observed. These results indicate that the 28,000-Da protein is covalently bound to the 5′ termini of the DNA.     

Isolation and purification of a polymeric form of the glycoprotein of rabies virus.

Of the three major proteins associated with the rabies virus membrane, only the glycoprotein was found to be located on the external surface of the virus membrane. Glycoprotein prepared by treatment of rabies virus with Triton X-100 and purified by isoelectric focusing was found to be homogeneous with respect to size and isoelectric point. This material, which is free of phospholipids, is able to protect in vaccination experiments against a lethal challenge infection with rabies virus. The apparent mol. wt. of this component isolated under non-denaturing conditions is approx. 400000. The same material analysed by SDS polyacrylamide gel electrophoresis (PAGE) was found to consist solely of polypeptide chains of the G protein (mol. wt. 80000). A minor glycoprotein (gp 50), detected by PAGE of the Triton X-100 released material, appeared to be a breakdown product of the G-protein. Therefore the detergent released material represents homopolymers of the G-protein. Whether the antigenic determinants reside on the monomeric subunit or are a property of the polymeric form of the G-protein is discussed.     

Antibodies specific for the phi 29 terminal protein inhibit the initiation of DNA replication in vitro

The phi 29 DNA-terminal protein serves as a primer for the initiation of DNA replication by covalently binding the first nucleotide in the DNA chain. Two distinct antibodies were used for functional analysis of this protein. One antibody was raised against sonicated phi 29 DNA-protein complex isolated from phage virions (anti-TP). The other antibody was raised against a conjugate of bovine serum albumin and a synthetic peptide corresponding to the carboxy-terminal of the phi 29 terminal protein (anti-gp3C), which was predicted from the nucleotide sequence of phi 29 DNA. Both antibodies react with native phi 29 terminal protein as determined by immunoprecipitation and enzyme-linked immunosorbent assay. Both antibodies specifically inhibit the complex-forming reaction between the phi 29 terminal protein and dAMP, the first nucleotide of phi 29 DNA.     

Membrane protein alterations in the neutrophil in response to chemotactic factors

The effect of chemotactic factors on surface membrane proteins of rabbit neutrophils was studied using lactoperoxidase-catalyzed iodination. Following external labeling, cells were dissociated with SDS and the soluble products analyzed by SDS polyacrylamide gel electrophoresis and radioautography. The chemotactic factors N-formyl-methionyl-Ieucyl-phenylalanine (FMLP) and C5a, at optimal doses for chemotaxis, induced the appearance of a new surface membrane associated protein band (mol. wt ～ 54,000). When cells were stimulated for optimal degranulation with cytochalasin B(CB) in the presence of FMLP or C5a, or with the calcium ionophore A23187, the appearance of the new band was intensified. In addition, the disappearance of a membrane associated protein band (mol wt ～ 44,000) was observed with cells stimulated for optimal degranulation. These protein bands appear to be associated with the cell surface membrane since: (1) the proteins could be removed by treatment of the cells with trypsin; and (2) most of the radioactivity was associated with purified cell membrane fractions. These results suggest that complex changes in surface membrane proteins occur in response to chemotactic and/or degranulating stimuli.     

Study of a new strain of paramyxoviruses isolated from wild ducks: structural polypeptides.

By polyacrylamide gel electrophoresis, Duck/Mississippi/75 virus was shown to contain five different types of polypeptides of mol. wt. ranging from 76 X 1O(3) to 43 X 10(3), two of which were glycosylated (mol. wt. 76 X 10(3) and 57 X 10(3). A comparison of this data with similar results obtained using Yucaipa and Newcastle disease virus (NDV) revealed a similarity with NDV, concerning the number, position and mol. wt. of the polypeptides. Haemagglutinating and neuraminidase properties are associated with surface glycoproteins which represent at least 40% of the virus protein. After KCl and Triton X-100 treatment and centrifugation on linear sucrose density gradients (10 to 25%, w/w) it was possible to isolate glycoprotein VP1 of mol. wt 76 X 10(3) with which haemagglutinating and neuraminidase activities were associated.