Probing DNA polymerase alpha with monoclonal antibodies

DNA polymerase alpha was purified from human KB cells by immunoaffinity chromatography. Enzyme activity was inhibited by three different monoclonal antibodies (SJK-132, SJK-211, SJK-287). Kinetic analysis showed that each antibody neutralized polymerase activity by a different mechanism. SJK-132 was competitive with DNA indicating it interacts with the DNA binding domain of the polymerase. SJK-287 showed a biphasic response to dCTP suggesting two dCTP binding sites exist on polymerase alpha. SJK-211 was non-competitive with DNA, dCTP and dATP.     

DNA denaturation for ultrastructural banding and the mechanism underlying the fluorochrome-photolysis-Giemsa technique studied with anti-5-bromodeoxyuridine antibodies

G- and R-bands produced by an immunochemical approach were studied by electron microscopy (EM) to evaluate the role of DNA denaturation on banding quality. Excelent banding was observed only after adequate denaturation by HCl, NaOH and formamide, used in appropriate concentrations to provide uniform 5-bromodeoxyuridine (BrdUrd) exposure by generating single-stranded DNA. Formamide treatment resulted in less intercellular variability. High temperature and high concentrations of NaOH and HCl altered chromosomal morphology. Besides formamide, Hoechst 33258 prestaining which does not interfere with the binding of the anti-BrdUrd antibody and UV irradiation associated with formamide also produced high quality banding. On the other hand, consecutive Hoechst and UV treatment completely inhibited the immunochemical banding. The data indicate that Hoechst and UV act synergistically to disintegrate BrdUrd-substituted chromatin from which DNA is then extracted, leaving only the unsubstituted DNA stainable with Giemsa.     

The molecular organization of human alpha 2-macroglobulin. An immunoelectron microscopic study with monoclonal antibodies

To study the three-dimensional organization of alpha 2-macroglobulin (alpha 2M) from human plasma, immunoelectron microscopy of negatively stained specimens was used. A panel of monoclonal antibodies (mAb) with specificities typical for the two major conformers of alpha 2M (native and protease-transformed) was explored. The mAb have been selected and were classified biochemically as specific for either native or transformed alpha 2M or as reactive with both conformers. Furthermore, among the mAb that were specific for the proteinase-transformed form of alpha 2M, those reacting with the 20-kDa receptor-binding domain were considered a fourth category. Immunoelectron microscopy with these 20-kDa receptor-binding domain-specific mAb yielded the most typical result: predominantly, individual H-like alpha 2M-chymotrypsin molecules were complexed with two IgG molecules, each one bound to the extremities of two arms of the H-like figure. The resulting planar complex has the appearance of a dumbbell. Since this was observed with eight different mAb of this specificity, the result is interpreted to mean that the 20-kDa receptor-binding domain is compact and constitutes the outermost domain at the extremes of the arms of the H-like transformed alpha 2M. The mAb which are specific for the transformed state of alpha 2M but which do not react with the 20-kDa receptor-binding domain, also bound at the arms of the H-like figure, but at nonterminal positions. Moreover, these mAb produced mostly linear, chain-like immune complexes of numerous H-like alpha 2M molecules cross-linked by the IgG. The large category of mAb that reacted with both conformers of alpha 2M (native and proteinase complex) were observed to make various types of immune complexes with intra- and intermolecular cross-linking by the IgG. The observations of reaction of these mAb with Cd2+-induced dimers (half-molecules of alpha 2M), either native or transformed, proved helpful and, for certain mAb, essential to understand the organization of the alpha 2M-IgG complexes. Combined, the observations allow us to propose new models for the three-dimensional organization of native and chymotrypsin-transformed dimeric and tetrameric human alpha 2M.     

Molecular cloning of DNA complementary to rat alpha 2-macroglobulin mRNA

Rat alpha 2-macroglobulin (alpha 2M) is an acute-phase protein synthesized in the liver. Using an in vitro translation system coupled with solid-phase radioimmunoassay, alpha 2M mRNA activity was found to rise to a maximum level in 16-24 h after turpentine injection. Poly(A)+ RNA from turpentine-injected rat liver was converted to cDNA by the method of Okayama-Berg, and about 50,000 transformants were obtained. From these transformants, clones containing alpha 2M cDNA were selected using the following criteria: 1) alpha 2M cDNA should hybridize with synthetic oligonucleotides encoding portions of the alpha 2M amino acid sequence, 2) alpha 2M cDNA should hybridize preferentially with RNA which increases during inflammation, 3) mRNA which hybridizes with alpha 2M cDNA should encode a polypeptide which specifically reacts with antibody against alpha 2M, and 4) the cDNA should contain the nucleotide sequences encoding the amino acid sequences of alpha 2M. We found clones which fulfilled these criteria. Using the cDNA clone as a probe, we demonstrated that the level of alpha 2M mRNA in the liver of inflamed animal markedly increased up to 1000-fold. The size of the alpha 2M mRNA was about 4800 nucleotides in length by Northern analysis.     

Non-random incorporation of 5-bromodeoxyuridine in rat cell DNA

Secondary cultures of rat embryo cells were exposed for 24 hrs. to 10-⁷M [³H] thymidine (TdR) or 10^?7M [³H]5-bromodeoxyuridine (BrdU) in order to localize and compare the distribution of the isotopes in DNA. DNA was extracted, sheared, and centrifuged to equilibrium through neutral and alkaline CsCl density gradients. The DNA band from each gradient type was separated into a “heavy” and “light” fraction, and DNA-DNA reassociation hybridizations were performed on each sample. Renaturation profiles revealed that each fractionated DNA sample was representative of the complete rat cell genome, except for the “light” [³H]BrdU-DNA prepared by centrifugation through alkaline CsCl gradients. This fraction was predominantly depleted of labeled late repetitive and intermediate sequences. Uncentrifuged rat DNA was sequentially fractionated during reassociation into rapidly, intermediate, and slowly reassociating sequences by hydroxyapatite chromatography. Relative specific activities of each component revealed a non-uniform distribution of [³H]BrdU moieties as compared to [³H]TdR. These results suggest a nonrandom incorporation of 10^?7M BrdU into rat cell DNA sequences.     

Properties and applications of new monoclonal antibodies raised against calf DNA polymerase alpha

A panel of 12 hybridoma cell lines secreting monoclonal antibodies against alpha-polymerase were prepared by fusion of mouse myeloma cells and spleen cells of a rat immunized with homogeneous calf thymus alpha-polymerase. Hybridomas were selected and cloned on the basis of immunobinding to pure alpha-polymerase in solid phase radioimmunoassay. Antibodies secreted by these cells eventually were purified in milligram quantities from ascites fluids. These antibodies, all of the rat immunoglobulin M class, cross-reacted with alpha-polymerases from calf and monkey cells as revealed by immunobinding in radioimmunoassay and by immunoprecipitation of DNA polymerase activity. The antibodies were not capable of neutralizing the enzyme activity. With the methods described these antibodies may be used to immunoprecipitate alpha-polymerase from crude extracts of mammalian cells and to measure levels of the enzyme protein.     

Immunocytochemical identification of the human alpha 2-macroglobulin receptor in monocytes and fibroblasts: monoclonal antibodies define the receptor as a monocyte differentiation antigen

The alpha 2-macroglobulin receptor was recently purified from rat liver and human placenta. Three different monoclonal antibodies have now been raised against the human receptor and expression of the 440-kDa receptor protein is demonstrated in human placenta, fibroblasts, liver, and monocytes by immunoblot analysis. Flow cytometric studies showed that anti-alpha 2-macroglobulin receptor monoclonal antibodies bind to 90-100% of the blood monocyte population and not to other blood cells. This defines the alpha 2-macroglobulin receptor as a monocyte differentiation antigen, different from any of the classified leucocyte cluster determinants. Electron microscopic gold immunocytochemistry revealed the subcellular distribution of the receptor in human cultured monocytes and fibroblasts. In these cells, 18-33% of the gold particles were found on the outside of the plasma membrane, and in fibroblasts, especially, in coated invaginations. The intracellular receptors were mainly distributed in vesicles and tubular structures.     

Incorporation of 5-bromodeoxyuridine into DNA in newborn rat tissues

The incorporation of bromodeoxyuridine (BUdR) into newborn rat tissue DNA has been determined after i.p. injection of 5-bromo-2′-deoxy[6-³H]uridine. Incorporation of the unchanged nucleoside was shown by hydrolysis and ion exchange chromatography of extracted DNA. In all tissues examined, more than 90% of the radioactivity incorporated was in the form of bromodeoxyuridine.     

Detection of 5-bromodeoxyuridine (BrdUrd) incorporation by monoclonal antibodies: role of the DNA denaturation step

Immunochemical detection of cells that incorporate 5-bromodeoxyuridine (BrdUrd) requires prior denaturation of DNA in situ to make BrdUrd binding sites accessible to the antibodies. A technique is described in which the DNA denaturation step is facilitated by a) prior dissociation of histones from DNA and b) the use of low ionic strength buffer in which the cells are suspended during heating. Dissociation of histones is achieved by cell treatment with 0.08N HCl at 0 degree C, which a) increases accessibility of DNA to propidium iodide (and following the denaturation to the antibodies); b) lowers stability of DNA to thermal denaturation; c) decreases differences between various cell types due to variability in chromatin structure; and d) ensures more complete DNA denaturation. Cell heating (80-95 degrees C) at low ionic strength (1 mM Na+) eliminates the need for formamide and results in extensive and rapid DNA denaturation. The method was applied in Friend leukemia, L1210 and HL-60 cell lines, and to bone marrow, experimental animal tumor and primary human tumor cells.     

Evaluation of S phase synchronization by analysis of DNA replication in 5-bromodeoxyuridine

The S phase kinetics have been evaluated in cells synchronized with either thymidine or hydroxyurea by direct analysis of the proportion of DNA semi-conservatively replicated as a function of time after release from the inhibitor. The proportion of DNA replicated was determined by growing the cells in medium containing 5-bromodeoxyuridine (BUdR) and subsequently measuring the amount of DNA that acquired increased buoyant density in CsCl gradients. The results confirm previous reports that substantial DNA synthesis occurs during TdR treatment. In contrast, HU provided a population of cells very nearly at the G 1-S interphase since 95 % of the DNA replicated synchronously after its removal. It is proposed that by measuring the rate and maximum extent of DNA replication with BUdR during S phase one can evaluate different synchrony methods for use in experiments designed to study aspects of semiconservative DNA replication.     

Calmodulin-specific monoclonal antibodies inhibit DNA replication in mammalian cells.

The involvement of calmodulin in the proliferation of Chinese hamster embryo fibroblast cells has been studied with a specific monoclonal antibody to calmodulin. We observed that calmodulin levels increase 2-fold in the late G1 period in these cells, and this coincides with the increase in DNA polymerase alpha activity as the cells progress synchronously from a quiescent state in the G1 to the S phase. However, there is a concurrent 10-fold enhancement of thymidine kinase activity, which is tightly coupled to the entry of cells into the S phase. Incubation of permeabilized S-phase cells with calmodulin-specific murine monoclonal antibody resulted in a dose-dependent inhibition of DNA replication. This inhibitory effect of anti-calmodulin antibodies on DNA replication is completely reversed by the addition of exogenously purified calmodulin. These observations provide evidence for the involvement of calmodulin in DNA replication and, therefore, in cell proliferation during the S phase.     

Isolation and purification of rat acute-phase alpha 2-macroglobulin

Acute-phase alpha 2-macroglobulin was highly purified from the serum of rats in which this protein had been induced 48 h previously by the injection of croton oil, an inflammatory agent. The isolation protocol involved two non-denaturing steps; first, separation according to molecular weight by gel filtration on Ultrogel AcA 22 and second, negative affinity chromatography which bound contaminating proteins to the column while allowing acute-phase alpha 2-macroglobulin to pass through. Several criteria were used to assess the purity of acute-phase alpha 2-macroglobulin, after which the protein by mass determination and by two different protein assays. Pure rat acute-phase alpha 2-macroglobulin was used to produce a monospecific antiserum and to calibrate a secondary standard of rat acute-phase serum by developing and characterizing rocket immunoelectrophoresis assay.     

Immunoelectron microscopy studies with a monoclonal antibody directed against a receptor recognition site epitope in human alpha 2-macroglobulin.

Alpha 2-Macroglobulin (alpha 2M) is a plasma proteinase inhibitor that binds up to 2 mole of proteinase per mole of inhibitor. Proteinase binding or reaction with small primary amines causes a major conformational change in alpha 2M. As a result of this conformational change, a new epitope recognized by monoclonal antibody 7H11D6 is exposed. The association of alpha 2M-proteinase or alpha 2M-methylamine with alpha 2M cellular receptors is prevented by 7H11D6. In this investigation, the binding of 7H11D6 to alpha 2M was studied by electron microscopy. 7H11D6 bound to alpha 2M-methylamine and alpha 2M-trypsin but not to native alpha 2M. The structure of alpha 2M after conformational change resembled the letter "H." 7H11D6 epitopes were identified near the apices of the four arms in the alpha 2M "H" structure. 7H11D6 that was adducted to colloidal gold (7HAu) retained the specificity of the free antibody (binding to alpha 2M-trypsin but not to native alpha 2M). alpha 2M conformational change intermediates prepared by sequential reaction with a protein crosslinker and trypsin also bound 7HAu. These results suggest that a complete alpha 2M conformational change is not necessary for 7H11D6 epitope exposure and may not be required for receptor recognition. 7HAu was used to isolate a preparation consisting primarily of binary alpha 2M-trypsin (1 mole trypsin per mole alpha 2M instead of 2). Structures resembling the letter "H" were most common; however, each field showed some atypical molecules with arms that were compacted instead of thin and elongated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental expression of the embryonic chicken brain DNA polymerase alpha and its binding with monoclonal antibodies against human KB cell DNA polymerase alpha.

Changes in DNA polymerase alpha activity accompanying tissue development have been well established in several systems. In most cases, DNA polymerase alpha activity decreases with development. Here, we report observed changes in DNA polymerase alpha activity throughout embryonic chicken brain (ECB) development. The level of DNA polymerase alpha activity was found to gradually decrease by 60% (2.3 to 0.8 nmol of [3H]dCMP incorporated/mg protein/h) between 9- and 19-day-old ECB. An enzyme-linked immunosorbent assay of DNA polymerase alpha utilizing monoclonal antibody SJK 237-71 (human KB cell DNA pol-alpha binder) also demonstrated a gradual decrease (up to 60%) of antigen over this same range of development. Analysis of DNA polymerase alpha from 11- and 19-day-old ECB by a 10 to 30% glycerol density gradient revealed a high molecular weight peak sedimenting near catalase (11.3 S) with activity at the 11th day being approximately 3-fold greater than activity at the 19th day. A Western immunoblot analysis utilizing monoclonal antibody SJK 237-71 (against human KB cell DNA polymerase alpha) showed a decrease in DNA polymerase alpha from 186 kilodaltons in 9- and 11-day ECB cell-free extracts to 120 kilodaltons in extracts from 13- to 19-day ECB. The conversion of DNA polymerase alpha from a higher to a lower molecular weight form may be a regulatory mechanism in eukaryotic DNA replication.     

Monoclonal antibodies against a glycoprotein localized in coated pits and endocytic vesicles inhibit alpha 2-macroglobulin binding and uptake

Eight monoclonal antibodies, all IgG2a, which recognize a 180/90-kDa glycoprotein similar in properties to the receptor for alpha 2-macroglobulin of mouse embryo 3T3 cell plasma membranes, have been tested for their effect on the binding and uptake of alpha 2-macroglobulin by live cells. One antibody directly inhibited binding of 125I-alpha 2-macroglobulin under conditions in which 125I-transferrin binding to the transferrin receptor was unaffected. Another monoclonal antibody decreased alpha 2-macroglobulin binding when preincubated with cells at 37 degrees C. This antibody was also capable of specifically binding to ligand-receptor complexes formed by preincubating 125I-alpha 2-macroglobulin with detergent extracts of Swiss 3T3 cells. Immunoelectron microscopy showed that the 180/90-kDa glycoprotein was localized in coated pits of the cell surface and in intracellular endocytic vesicles (receptosomes/endosomes). The data suggest that the 180/90-kDa glycoprotein is a component of the receptor for alpha 2-macroglobulin.     

Immunological and biochemical differentiation of guanyl nucleotide binding proteins: interaction of Go alpha with rhodopsin, anti-Go alpha polyclonal antibodies, and a monoclonal antibody against transducin alpha subunit and Gi alpha

Guanyl nucleotide binding proteins couple agonist interaction with cell-surface receptors to an intracellular enzymatic response. In the adenylate cyclase system, inhibitory and stimulatory effects are mediated through guanyl nucleotide binding proteins, Gi and Gs, respectively. In the visual excitation complex, the photon receptor rhodopsin is linked to its target, cGMP phosphodiesterase, through transducin (Gt). Bovine brain contains another guanyl nucleotide binding protein, Go. The proteins are heterotrimers of alpha, beta, and gamma subunits; the alpha subunits catalyze receptor-stimulated GTP hydrolysis. To examine the interaction of Go alpha with beta gamma subunits and rhodopsin, the proteins were reconstituted in phosphatidylcholine vesicles. The GTPase activity of Go alpha purified from bovine brain was stimulated by photolyzed, but not dark, rhodopsin and was enhanced by bovine retinal Gt beta gamma or by rabbit liver G beta gamma. Go alpha in the presence of G beta gamma is a substrate for pertussis toxin catalyzed ADP-ribosylation; the modification was inhibited by photolyzed rhodopsin and enhanced by guanosine 5'-O-(2-thiodiphosphate). ADP-Ribosylation of Go alpha by pertussis toxin inhibited photolyzed rhodopsin-stimulated, but not basal, GTPase activity. It would appear from this and prior studies that Go alpha is similar to Gt alpha and Gi alpha; all three proteins exhibit photolyzed rhodopsin-stimulated GTPase activity, are pertussis toxin substrates, and functionally couple to Gt beta gamma. Go alpha (39K) can be distinguished from Gi alpha (41K) but not from Gt alpha (39K) by molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of monoclonal antibodies directed against bromodeoxyuridine with pyrimidine bases, nucleosides, and DNA

Although antibodies directed against bromodeoxyuridine (BrdU) are being used in both clinical and basic research laboratories as tools to study and monitor DNA synthesis, little is known about the epitopes with which they react. Four monoclonal antibodies directed against BrdU were produced and were characterized to learn more about the epitopes on BrdU which are important for antibody recognition, to identify compounds other than BrdU which react with the antibodies and which might interfere with immunologic assays for BrdU, and to characterize the reaction of these antibodies with BrdU-containing DNA. By radioimmunoassays, the antibodies generally reacted well with 5-iododeoxyuridine, 5-fluorodeoxyuridine, and 5-nitrouracil. However, none of the antibodies reacted well with uridine--indicating that a substituent on uridine C5 was essential for antibody reactivity--or with 5-bromo- or iodo-cytosine, indicating that the region around pyrimidine C4 is important for antibody recognition. Although the antibodies reacted with 5-halogen-substituted uracil bases, the antibodies reacted much better with the corresponding halogenated nucleosides, indicating that the sugar moiety was important for recognition. The presence of a triphosphate group on C'5 of BrdU (i.e., BrdUTP) did not detectably alter antibody recognition. Three of the antibodies reacted only with purified DNA containing BrdU, whereas one antibody, which exhibited a weak interaction with thymidine, also reacted with BrdU-free DNA. S1 nuclease treatment of purified DNA suggested that all four monoclonal antibodies reacted exclusively with single-stranded regions of BrdU-containing DNA. Comparison of detecting DNA synthesis by [3H]TdR incorporation followed by autoradiography with that by BrdU incorporation followed by indirect immunofluorescence indicated that the latter technique was both an accurate and a sensitive measure of DNA synthesis.     

Enzymatic determination of nonrandom incorporation of 5-bromodeoxyuridine in rat DNA.

Secondary cultures of normal rat embryo cells were synchronized by a double thymidine block and pulsed with 10(-7) M 5-[3H]bromodeoxyuridine (BrdUrd) OR 10(-7) M[3H]thymidine during an entire S phase (7.5 h). To examine the pattern of [3H]thymidine, DNA was immediately extracted and purified at the completion of the S phase, CsCl density gradient centrifugation revealed that substitution for thymine by bromouracil was less than 7%. Single-strand specific nucleases obtained from Aspergillus oryzae and Neurospora crassa were allowed to react with native and partially depurinated (24-29%) [3H]BrdUrd-labeled rat DNA samples, and the products were assayed by hydroxylapatite column chromatography. Approximately 4-6% of the native, nondepurinated rat DNA was hydrolyzed by both nucleases. However, 24-28% of the partially depurinated, [3H] thymidine-labeled rat DNA was hydrolyzed by both enzymes as determined by loss of mass as well as radioactivity. Whereas comparable levels of depurinated, [3H]BrdUrd-labeled DNA were physically hydrolyzed by both nucleases, nearly 65% of the radioactivity was not recovered. Native, as well as depurinated, enzyme-treated DNA samples were sequentially and preparatively reassociated into highly repetitive, middle repetitive, and nonrepetitive nucleotide sequence components. The absolute and relative specific activities of each subfraction of native [3H]thymidine-labeled DNA were comparable. [3H]BrdUrd was differentially concentrated in the middle repetitive sequences as compared to other reiteration frequency types. When depurinated, nuclease-treated DNA samples were similarly fractionated, [3H]thymine moieties were uniformly distributed thoughout all sequences. However, a differential loss of [3H]BrdUrd moieties was detected predominantly from the middle repetitive nucleotide fraction. Melting profiles of the renatured DNA samples were characteristic of each respective DNA subfraction regardless of isotopic precursor. These results suggest that [3H]BrdUrd may be differentially incorporated into A + T rich clusters of rat DNA, especially in the moderately repeated chromosomal elements.