Methionine-labeled Cell Extracts Research Articles

Identification of the alpha6 integrin as a candidate receptor for papillomaviruses.

Papillomaviruses (PVs) bind in a specific and saturable fashion to a range of epithelial and other cell lines. Treatment of cells with trypsin markedly reduces their ability to bind virus particles, suggesting that binding is mediated via a cell membrane protein. We have investigated the interaction of human PV type 6b L1 virus-like particles (VLPs) with two epithelial cell lines, CV-1 and HaCaT, which bind VLPs, and a B-cell line (DG75) previously shown not to bind VLPs. Immunoprecipitation of a mixture of PV VLPs with [35S]methionine-labeled cell extracts and with biotin-labeled cell surface proteins identified four proteins from CV-1 and HaCaT cells of 220, 120, 87, and 35 kDa that reacted with VLPs and were not present in DG75 cells. The alpha6beta4 integrin complex has subunits corresponding to the VLP precipitated proteins, and the tissue distribution of this complex suggested that it was a candidate human PV receptor. Monoclonal antibodies (MAbs) to the alpha6 or beta4 integrin subunits precipitated VLPs from a mixture of CV-1 cell proteins and VLPs, whereas MAbs to other integrin subunits did not. An alpha6 integrin-specific MAb (GoH3) inhibited VLP binding to CV-1 and HaCaT cells, whereas an anti-beta4 integrin MAb and a range of integrin-specific and other MAbs did not. Furthermore, human laminin, the natural ligand for the alpha6beta4 integrin, was able to block VLP binding. By use of sections of monkey esophagus, the distribution of alpha6 integrin expression in the basal epithelium was shown to coincide with the distribution of bound VLPs. Taken together, these data suggest that VLPs bind specifically to the alpha6 integrin subunit and that integrin complexes containing alpha6 integrin complexed with either beta1 or beta4 integrins may act as a receptor for PV binding and entry into epithelial cells.

Changes in translatable mRNA species associated with nutrient deprivation and protease synthesis in Metarhizium anisopliae

Summary: We have studied the regulation of the extracellular chymoelastase protease (PrI) of the fungal entomopathogen Metarhizium anisopliae. This enzyme is involved in the penetration of insect cuticle by Metarhizium and other entomopathogenic fungi. Changes in mRNA sequences during starvation-induced synthesis of chymoelastase were investigated by comparing poly(A+)RNAs and their complementary DNA in rapidly growing or nutrient-deprived cultures of Metarhizium. Hybrid-selected translation revealed that three novel polypeptides (41, 40·2 and 29·8 kDa) were produced rapidly (<1 h) during nutrient deprivation; the most intensely translated species (41 kDa) was identified as the primary translation product of Pr1 by examination with anti-Pr1-IgG. Measurement of starvation-specific RNA levels by dot-blot hybridization with [32P]cDNA showed that transcripts were not present in spores but were produced when the formation against a hard surface of infection structures was induced by depletion of endogenous nutrient reserves. Starvation-specific mRNA species failed to appear when new RNA synthesis was blocked with actinomycin D, indicating that control is at the level of transcription. Immunoblot analysis with anti-Pr1-IgG showed that Pr1 protein increased in concert with the appearance of Pr1 transcripts and active enzyme. Two very short lived precursors of Pr1, the primary translation product (41 kDa) and an intermediate (40·8 kDa) in addition to mature Pr1 (30 kDa) were detected by immunoprecipitation of [35S]methionine-labelled cell extracts with anti-Pr1-IgG. Pulse-labelling experiments demonstrated that a time of about 7 min was required for [35S]methionine to be processed into extracellular Pr1. These results suggest that regulation of Pr1 gene expression occurs during starvation which, coupled with fast processing, allows very rapid secretion of Pr1. Isolates of four other entomopathogenic fungi (Verticillium lecanii, Beauveria bassiana, Tolypocladium niveum and Paecilomyces farinosus) produced Pr1-type enzymes during nutrient deprivation, suggesting our results may have widespread application in understanding entomopathogenicity.

Monoclonal autoantibody recognizing a unique set of small nuclear ribonucleoprotein complexes.

A murine IgG2a, kappa-monoclonal autoantibody (mAb) F78 is described that recognizes a novel epitope associated with small nuclear ribonucleoprotein complexes (snRNP). F78 selectively immunoprecipitated a unique pattern of small nuclear RNA (U1, U2, and U4 to U6) characterized by a marked depletion of U1 and an elevated proportion of U2 compared with known patterns immunoprecipitated by previously described anti-RNP (2.73) and anti-Sm (7.13, Y12) mAb. Analysis of immunoprecipitated RNA from extracts previously cleared with mAb F78 and probed with anti-RNP mAb 2.73 further indicated the presence of two distinct subsets of U1. Immunoblots of whole cell extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) without heating showed that F78 selectively bound to a trypsin-sensitive component of apparent m.w. greater than 120,000 which was decreased in size following RNase A treatment. The anti-Sm mAb, but not the anti-RNP mAb, also recognized this component in unheated samples. Heating before SDS-PAGE resulted in abrogation of binding to the F78 epitope. Immunoprecipitation of unlabeled or [35S]methionine-labeled cell extracts with F78 revealed the presence of most snRNP peptides, but the absence of peptide C and the 68,000 m.w. component, known to be selectively associated with U1-specific snRNP. Two-dimensional SDS-PAGE analysis of F78 immunoprecipitates confirmed that the epitope recognized by this mAb resides on a heat-dissociable complex containing snRNP-related peptides B, B', D, E, F, and G, but lacking U1-associated peptides. F78 mAb therefore defines a subset of snRNP which lack anti-RNP associated U1 RNA as well as peptides known to be selectively associated with this RNA species. It apparently recognizes an epitope associated with an assembled form of these particles and may be useful in examining structures involved in RNA processing.

Characterization of herpesvirus sylvilagus glycoproteins released into the culture medium of infected cells: antisera to gp13 and gp32 neutralize viral infectivity in vitro and identify antigens on plasma membranes of infected cells.

Polypeptides released into the culture medium of herpesvirus sylvilagus-infected cells were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracellular fluid from [35S]methionine- and [3H]glucosamine-labeled cell cultures. Virus-induced glycoproteins 31, 32, and 33 (molecular weights of 62,000, 59,000, and 54,000, respectively) were the most abundant species and appeared predominantly in the culture medium. This observation, together with the known cell-associated nature of herpesvirus sylvilagus, suggested that virus-induced glycoproteins 31, 32, and 33 were specifically released. Immunization of rabbits with virus-induced glycoproteins 13 (molecular weight of 130,000) and 32 resulted in the production of antibodies that neutralized viral infectivity in vitro. Both antiserum to gp13 and antiserum to gp32 immunoprecipitated gp13, gp26, gp33a, gp45, and virus-induced polypeptide 39 (molecular weights of 130,000, 77,000, 49,000, 27,000, and 36,000, respectively) from [35S]methionine-labeled cell extracts as well as virus-induced glycoproteins 31, 32, and 33 from the culture medium. In addition, membrane immunofluorescence assays indicate that an antigen(s) reactive with anti-gp13/32 serum was located on the plasma membrane of infected cells.

Processing and secretion of the Yarrowia lipolytica RNase.

Secretion of the extracellular RNase from the yeast Yarrowia lipolytica was studied in pulse-chase and immunoprecipitation experiments. A polypeptide of 45,000 daltons was immunoprecipitated from [35S]methionine-labeled cell extracts and supernatant medium by rabbit anti-RNase antiserum. The RNase was secreted rapidly; the time between synthesis and appearance in the extracellular medium was about 5 min. In pulse-chase experiments, about 50% of the RNase was still cell associated 30 min after labeling. A polypeptide of 73,000 daltons whose immunoprecipitation was blocked by an excess of purified RNase was also detected. It broke down to a polypeptide with the same mobility and same peptide map as the mature RNase. Peptide maps of the undegraded 73-kilodalton polypeptide and the intracellular mature RNase contained several peptides of identical mobility. Immunoprecipitates from cells labeled in the presence of tunicamycin contained 66- and 45-kilodalton polypeptides. Endoglycosidase H treatment of the 73-kilodalton polypeptide converted it to a 66-kilodalton form, but did not change the apparent molecular weight of the mature form of the RNase. Labeling kinetics from pulse-chase experiments did not clearly support a precursor-product relationship between the 73-kilodalton polypeptide and the intracellular 45-kilodalton form of the RNase, and other relationships between the two polypeptides are possible.

The low density lipoprotein receptor on human peripheral blood monocytes and lymphocytes: visualization by ligand blotting and immunoblotting techniques.

Western blotting and immunoprecipitation techniques were used to study low density lipoprotein (LDL) receptors from cultured human monocytes, lymphocytes, and fibroblasts. After incubation in lipoprotein-deficient media to allow induction, receptors were solubilized, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose paper, and detected by incubation with apolipoprotein B-containing lipoproteins followed by radiolabeled antiapolipoprotein B antibody. LDL receptors of identical apparent mol wt were demonstrated in monocyte and lymphocyte cell extracts; the receptors bound LDL as well as human post-prandial lipoprotein (density, less than 1.0063) on Western blots, but did not bind acetyl-LDL. LDL receptors in mononuclear cells could also be detected by immunoblotting using immunoglobulin G-C7, a monoclonal antibody raised against the bovine LDL receptor. When cell extracts were blotted, the mononuclear cell receptors had a lower mol wt than the fibroblast receptor in unreduced sodium dodecyl sulfate-polyacrylamide gels, with an apparent mol wt difference of 5,000 [134,000 +/- 1,400 (+/- SE) for mononuclear cells vs. 139,000 +/- 1600 for fibroblasts]. Immunoprecipitation and electrophoresis of L-[35S]methionine-labeled cell extracts revealed more rapid conversion of the receptor precursor to the mature receptor in monocytes than in fibroblasts. Western blotting of mononuclear cells from a patient with an abnormally high mol wt receptor characterized in fibroblasts demonstrated that the same mutation was expressed in monocytes and lymphocytes. This report represents the first visualization of human mononuclear cell LDL receptors by Western blotting and immunoprecipitation. These techniques may find application in population screening for LDL receptor variability.

Immunoprecipitation of the low-density-lipoprotein (LDL) receptor and its precursor from human monocyte-derived macrophages.

Synthesis of the low-density-lipoprotein (LDL) receptor protein by cultured human monocyte-derived macrophages was demonstrated by immunoprecipitation of [35S]methionine-labelled cell extracts with a monoclonal antibody to the bovine adrenal LDL receptor. Although the antibody does not bind to or inhibit binding of 125I-LDL to the LDL receptor on intact fibroblasts, it specifically binds to a protein in extracts of human skin fibroblasts, of Mr approx. 130,000 under non-reducing conditions, that is able to bind LDL. In monocyte-derived macrophages, as in fibroblasts, the receptor is synthesized as a low-Mr precursor that is converted into the mature protein. The half-life of the precursor in human macrophages is approx. 44 min. In cells from two homozygous familial-hypercholesterolaemic subjects, only the precursor form of the receptor is synthesized. Detection of abnormalities of LDL-receptor synthesis in human mononuclear cells may be a useful aid in diagnosis of familial hypercholesterolaemia that is simpler and quicker than methods requiring growth of cultured skin fibroblasts.

Intracellular synthesis of human parainfluenza type 3 virus-specified polypeptides.

The intracellular synthesis of human parainfluenza type 3 virus-specified polypeptides was examined by polyacrylamide gel electrophoresis of [35S]methionine-labeled cell extracts under reducing conditions. All of the virion structural proteins were detected in cell extracts, including: L, 180,000 molecular weight (180K); P, 83K; HN, 69K; NP, 66K; F0, 60K; F1, 51K; and M, 38K. P and NP were phosphorylated. HN and F were glycosylated. The kinetics of intracellular viral protein synthesis did not detect any early or late proteins. Pulse-chase experiments failed to detect any precursor-product relationships. No nonstructural proteins were detected.

Identification of herpesvirus sylvilagus-induced polypeptides in productively infected cells.

Polypeptides synthesized in cell cultures infected with high multiplicities of herpesvirus sylvilagus were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled cell extracts. Initiation of polypeptide synthesis was detected by 6 h after infection. The maximum intensity of many [35S]methionine-labeled viral bands was observed at 45 h after infection. Production of detectable infectious virus began between 18 and 24 h and reached a plateau at 48 h after infection. Immunoprecipitation of cell extracts identified a minimum of 45 virus-induced polypeptides ranging in molecular weight from 230,000 to 27,000. The major polypeptide appeared to have a molecular weight of 150,000. The pattern of these extracts suggested that the synthesis of host polypeptides is stimulated during the first 12 h and thereafter reduced, but not completely inhibited, during the remaining course of infection.

Effect of microtubule assembly status on the intracellular processing and surface expression of an integral protein of the plasma membrane.

We studied the effects of changes in microtubule assembly status upon the intracellular transport of an integral membrane protein from the rough endoplasmic reticulum to the plasma membrane. The protein was the G glycoprotein of vesicular stomatitis virus in cells infected with the Orsay-45 temperature-sensitive mutant of the virus; the synchronous intracellular transport of the G protein could be initiated by a temperature shift-down protocol. The intracellular and surface-expressed G protein were separately detected and localized in the same cells at different times after the temperature shift, by double-immunofluorescence microscopic measurements, and the extent of sialylation of the G protein at different times was quantitated by immunoprecipitation and SDS PAGE of [35S]methionine-labeled cell extracts. Neither complete disassembly of the cytoplasmic microtubules by nocodazole treatment, nor the radical reorganization of microtubules upon taxol treatment, led to any perceptible changes in the rate or extent of G protein sialylation, nor to any marked changes in the rate or extent of surface appearance of the G protein. However, whereas in control cells the surface expression of G was polarized, at membrane regions in juxtaposition to the perinuclear compact Golgi apparatus, in cells with disassembled microtubules the surface expression of the G protein was uniform, corresponding to the intracellular dispersal of the elements of the Golgi apparatus. The mechanisms of transfer of integral proteins from the rough endoplasmic reticulum to the Golgi apparatus, and from the Golgi apparatus to the plasma membrane, are discussed in the light of these observations, and compared with earlier studies of the intracellular transport of secretory proteins.

Induction of cytochrome P-450 isozymes in rat hepatoma-derived cell cultures.

We have investigated the responsiveness of established rat hepatocyte cell cultures to inducers of cytochrome P-450. One Reuber hepatoma-derived line (Fu5-C8), which under normal culture conditions produces no detectable cytochrome P-450(MC) or cytochrome P-450(PB)--the major cytochrome P-450 isozymes induced by 3-methylcholanthrene and phenobarbital, respectively--was tested for the ability to accumulate either cytochrome P-450 isozyme in response to treatment with various xenobiotics. By immune-precipitation from [35S]-methionine-labeled cell extracts, using monospecific anticytochrome P-450(MC) antibody or monoclonal anticytochrome P-450(PB) antibody, it was demonstrated that these cells possess the capability to synthesize cytochrome P-450(MC) in response to 3-methylcholanthrene treatment, while none of the drug treatments caused the synthesis of detectable quantities of cytochrome P-450(PB). RNA extracted from Fu5-C8 cells directed the in vitro synthesis of immune-precipitable cytochrome P-450(MC) only after treatment of the cells with 3-methylcholanthrene. Kinetic analysis of the response of these cells to 3-methylcholanthrene induction revealed detectable levels of immune-precipitable cytochrome P-450(MC) 2 h after drug treatment with maximal induction occurring between 12 and 16 h of exposure. Another cell line (HF 1.5), obtained originally by hybridization of Fao X H5 variants of a Reuber H35 hepatoma, produces cytochrome P-450(MC) and also cytochrome P-450(PB) constitutively, as determined by specific immune-precipitation from labeled cell extracts. Exposure of confluent monolayers to either phenobarbital or 3-methylcholanthrene resulted in an induction of cytochrome P-450(PB) or cytochrome P-450(MC), respectively. Double-labeling immunofluorescence studies indicate that all cells in the culture produce albumin and most of the cells produce cytochrome P-450(MC), but only a subset of cells synthesize cytochrome P-450(PB). Our results demonstrate that some continuously dividing hepatocyte cell cultures retain the capacity to respond to xenobiotics, including phenobarbital, a response which is typically exhibited by fully differentiated liver cells. Such established hepatocyte cell cultures should prove useful for investigating the mechanism of induction of cytochrome P-450(PB).

Down-regulation of the epidermal growth factor receptor in KB cells is due to receptor internalization and subsequent degradation in lysosomes.

Using a monoclonal antibody to the human epidermal growth factor (EGF) receptor (EGF-R1), we have followed the metabolism of the receptor and the pathway of its internalization in KB cells after the addition of EGF. Measurement of surface binding of 125I-labeled EGF showed that about 80% of EGF binding activity disappeared from the plasma membrane after a 10-min exposure to EGF at 37 degrees C. Immunoprecipitation of the receptor from [35S]methionine-labeled cell extracts with EGF-R1 showed that EGF caused the receptor to be degraded with a half-life of 40 min. Immunofluorescence using EGF-R1 showed an EGF-dependent redistribution of the EGF receptor. In cells not exposed to EGF, almost all of the receptor was diffusely distributed on the cell surface. After EGF addition, the receptor was rapidly internalized, first appearing in small punctate organelles characteristic of receptosomes and then in larger perinuclear lysosome-like structures. By 120 min almost all of the immunoreactive EGF receptor had disappeared from the cells. Immunocytochemistry at the electron microscopic level confirmed these light microscopic findings. The diffusely distributed receptor on the cell surface first clustered into clathrin-coated pits in the presence of EGF, next was internalized into receptosomes, appeared transiently in transreticular Golgi elements, and finally was seen in lysosomes. This EGF-dependent down-regulation and degradation of the EGF receptor in KB cells provides a striking example of ligand-dependent clustering and internalization of a receptor, followed by degradation in lysosomes of both ligand and receptor.

A functional simian virus 40 origin of replication is required for the generation of a super T antigen with a molecular weight of 100,000 in transformed mouse cells.

We used two recombinant plasmids, one containing wild-type simian virus 40 DNA (pSVR1) and the other containing a simian virus 40 genome with a defective origin of replication (pSVR1-origin-minus) to transfect NIH3T3 cells. Quantitation of T-antigen synthesis by indirect immunofluorescence at 48 h after transfection with either DNA revealed the same percentage of T-positive nuclei. The transformation frequencies observed were also similar with both plasmids. Immunoprecipitation of [35S]methionine-labeled cell extracts showed the expected 94,000-dalton (94K) T and 17K t antigens in all clones examined. In pSVR1-generated transformants, a 100K super T antigen was also detected. Transformants isolated from pSVR1-origin-minus transfection, however, never expressed this 100K super T antigen, and some of these clones originally also showed greatly reduced levels of 94K T antigen. However, after growth in culture for several generations, the levels of 94K T antigen synthesis in these underproducer clones were dramatically increased. A direct correlation between the amounts of T antigen synthesized and the ability to grow independently of anchorage was observed. The mechanism which brings about increasing levels of T-antigen synthesis in some of the clones is not clear, but it appears not to be due to changes in either the copy number or the methylation pattern of the integrated simian virus 40 DNA.

Isolation of a cell surface receptor protein for laminin from murine fibrosarcoma cells.

We used affinity chromatography to isolate a specific laminin-binding protein from murine fibrosarcoma cells. These cells bind exogenous laminin to their surface with high affinity (Kd = 2 X 10(-9)M for laminin) with approximately 5 X 10(4) sites per cell. Laminin affinity chromatography of [35S]methionine-labeled cell extracts produced two distinct proteins. One was identified as Type IV (basement membrane) collagen based on its migration pattern on SDS gels and bacterial collagenase sensitivity. The other protein, which migrates as a single band or closely spaced doublet on reduced SDS gels, has a reduced molecular weight of 69,000. Using a nitrocellulose filter disk assay, we found that the latter protein specifically bound 125I-laminin with the same high affinity (Kd = 2 X 10(-9)M for laminin) as did intact fibrosarcoma cells. By iodinating intact cells, we demonstrated that this laminin-binding protein is on the cell surface. We conclude that this protein with reduced molecular weight of 69,000 is a subunit or component of a larger cell surface receptor protein for laminin in this fibrosarcoma model. This laminin receptor may mediate the interaction of the cell with its extracellular matrix.

Characterization of polypeptides serologically and structurally related to hexosaminidase in cultured fibroblasts.

Human fibroblasts synthesize several polypeptides that assort into the various forms of hexosaminidase (hex). We report here the occurrence of three newly identified, hexosaminidase-related polypeptides resolved by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis of immunoprecipitates from [35S]methionine-labeled cell extracts. These polypeptides, called band 2 (75,000), band 3 (70,000), and band 4 (63,000), were immunoprecipitated by an antiserum specific to placental hex I2. They are distinct from pre-alpha- (60,000) and pre-beta- (58,000) precursor polypeptides and the alpha- (56,000), beta a- (27,000), and beta b- (27,000) polypeptides of the mature hex A (alpha beta a beta b) and hex B (2[beta a beta b]). When fibroblast extracts were chromatographed on DEAE-Sepharose, bands 2, 3, and 4 were eluted together in fractions before hex A, in a position characteristic of serum and placental hex I2 and serum hex P. This suggests that bands 2, 3, and 4 might represent the polypeptides of a fibroblast hex I. The analysis of partial proteolytic digests of the radioactively labeled polypeptides revealed that bands 2 and 3, pre-beta, and beta a had several peptides in common, suggesting that they are structurally related to each other. However, bands 2, 3, and 4 were present in extracts of Tay-Sachs (pre-alpha and alpha deficiency) and Sandhoff cells (pre-beta, beta a, and beta b deficiency) and appeared later than pre-beta in pulse-chase experiments. These results suggest that bands 2 and 3 occur independently of pre-beta and beta a and are probably specified by different mRNA, whether from the same gene or distinct but homologous genes.

Nonlytic simian virus 40-specific 100K phosphoprotein is associated with anchorage-independent growth in simian virus 40-transformed and revertant mouse cell lines.

Normal fibroblasts display two distinct growth controls which can be assayed as requirements for serum or for anchorage. Interaction of mouse 3T3 fibroblasts with simian virus 40 (SV40) thus generates four classes of transformed cells. We have examined viral gene expression in these four classes of cell lines. Immunoprecipitation of [35S]methionine-labeled cell extracts with an antiserum obtained from tumor-bearing hamsters detected the SV40 large T and small t proteins (94,000 molecular weight [94K], 17K) and the nonviral host 54K protein in all cell lines tested. A tumor antigen with an apparent molecular weight of 100,000 was also found in some, but not all, lines. Similar "super T" molecules have been found by others in many rodent transformed lines. We carried out an analysis of the relation of phenotype to relative amounts of these proteins in cell lines of the four classes, using the Spearman rank correlation test. The amount of the 100K T antigen relative to the 94K T antigen or to total viral protein was well correlated with the ability to form colonies in semisolid medium. No significant correlation was found between quantities of labeled 94K T antigen, 54K host antigen, or 17K t antigen and either serum or anchorage independence. Mouse cells transformed with the small t SV40 deletion mutant 884 synthesized a 100K T antigen, suggesting that small t is not required for the production of this protein. The 100K T antigen migrated more slowly than lytic T. Since mixtures of extracts from cells expressing and lacking the 100K T antigen yielded the expected amount of this protein, it is unlikely that the 100K T derives from the 94K protein by a posttranslational modification.

Nonlytic simian virus 40-specific 100K phosphoprotein is associated with anchorage-independent growth in simian virus 40-transformed and revertant mouse cell lines.

Normal fibroblasts display two distinct growth controls which can be assayed as requirements for serum or for anchorage. Interaction of mouse 3T3 fibroblasts with simian virus 40 (SV40) thus generates four classes of transformed cells. We have examined viral gene expression in these four classes of cell lines. Immunoprecipitation of [35S]methionine-labeled cell extracts with an antiserum obtained from tumor-bearing hamsters detected the SV40 large T and small t proteins (94,000 molecular weight [94K], 17K) and the nonviral host 54K protein in all cell lines tested. A tumor antigen with an apparent molecular weight of 100,000 was also found in some, but not all, lines. Similar "super T" molecules have been found by others in many rodent transformed lines. We carried out an analysis of the relation of phenotype to relative amounts of these proteins in cell lines of the four classes, using the Spearman rank correlation test. The amount of the 100K T antigen relative to the 94K T antigen or to total viral protein was well correlated with the ability to form colonies in semisolid medium. No significant correlation was found between quantities of labeled 94K T antigen, 54K host antigen, or 17K t antigen and either serum or anchorage independence. Mouse cells transformed with the small t SV40 deletion mutant 884 synthesized a 100K T antigen, suggesting that small t is not required for the production of this protein. The 100K T antigen migrated more slowly than lytic T. Since mixtures of extracts from cells expressing and lacking the 100K T antigen yielded the expected amount of this protein, it is unlikely that the 100K T derives from the 94K protein by a posttranslational modification.

Identification of polypeptide components of adenovirus type 12 tumor antigen from productively and abortively infected cells and from tumor cells

Adenovirus type 12 (Ad12) tumor antigen (T antigen) induced in productively infected KB cells, abortively infected rabbit RK13 cells, and hamster tumor cells (THA cells) was studied by immunoprecipitation and polyacrylamide gel electrophoresis. [ 35S]Methionine-labeled cell extracts were precipitated with anti-T serum, obtained from hamsters bearing tumors induced by Ad12, and with anti-P serum, obtained from rabbits immunized against Ad12 early proteins induced in RK13 cells. Two polypeptides with 31,000 (31K) and 19K molecular weights are precipitated from all cells. Polypeptides with 16K, 15K, 13K, and 10.5K molecular weights are observed in extracts from KB and THA cells only. T antigen from tumor cells also contains 57K, 50K, 45K, 41K, and 34K polypeptides. Polypeptides with 39K, 28K, and 25K molecular weights are synthesized in KB and RK13 cells, mainly when the cells are pretreated with cycloheximide, but are never seen in extracts from tumor cells. Anti-P serum precipitates the 39K, 31K, 28K, 25K, and 19K polypeptides and also a 60K polypeptide from KB and RK13 cells, which probably corresponds to the DNA-binding protein.

P53 transformation-related protein: detection by monoclonal antibody in mouse and human cells.

A transformation-related protein of M(r) 53,000, designated p53, has been detected in a range of neoplastic cell types of the mouse by using immunoprecipitation of [(35)S]-methionine-labeled cell extracts with mouse antiserum [DeLeo, A. B., Jay, G., Appella, E., DuBois, G. C., Law, L. W. & Old, L. J. (1979) Proc. Natl. Acad. Sci. USA 76, 2420-2424]. We have now prepared a monoclonal antibody to p53 and have used it to study the occurrence and intracellular location of p53 by indirect immunofluorescence assays. In accordance with the results of immunoprecipitation, these tests showed p53 in all 13 transformed mouse cell lines studied. In each case, p53 was found in the nucleus. No p53 was detected in normal mouse fibroblasts, 3T3 cells, bone marrow cells, thymus cells, or embryo cells. A serologically related protein was detected in the nucleus of human cells by monoclonal antibody and was found in both normal and neoplastic cultured cells. Expression of p53 in human cells correlates with the growth characteristics of the culture, high p53 levels being associated with rapid cell proliferation and low p53 levels, with cessation of cell division. Normal and malignant human cells differ, however, with regard to the effect of confluency on p53 expression. Normal kidney epithelium and fetal brain cells, which express high p53 levels during exponential growth, show a prompt decrease in p53 associated with contact inhibition of cell division. Malignant cells, on the other hand, continue to express p53 after confluency and subsequent overgrowth of the monolayers. These results suggest that p53 may be involved in the normal regulation of cell division and that malignant transformation leads to abnormalities in the control of p53 expression.

Analysis of HLA-D region-associated molecules with monoclonal antibody.

A group of monoclonal antibodies directed against nonpolymorphic HLA-D antigens (human Ia antigens) was produced by somatic cell hybridization. One of these antibodies was used to analyze the two-dimensional polyacrylamide gel electrophoretic pattern of the HLA-D alloantigens from different HLA-D homozygous B-cell lines. Two-dimensional gels of [35S]methionine-labeled cell extracts immunoprecipitated by a monoclonal antibody directed against HLA-D antigens showed a complex set of spots whose electrophoretic pattern varied according to the HLA-D type. The major HLA-D-related electrophoretic polymorphism was found in the basic and smaller (26,000-28,000 daltons) HLA-D polypeptides. These patterns represent allele-specific "fingerprints" of different HLA-D genotypes. There are striking similarities with respect to number, size, and charge spectrum among HLA-D polypeptides and the murine I-A and I-E subregion polypeptides, suggesting a similar genetic organization and molecular complexity in both species.