phosphorus-radioisotopes and Cell-Transformation--Viral

Two site-specific anti-peptide antisera have been produced that efficiently recognize the native form of the v-erb B protein from avian erythroblastosis virus (AEV)-infected chicken erythroblasts and fibroblasts, and from AEV-transformed mammalian cells. Since the antibodies were generated against synthetic sequences, the immunoprecipitations could be performed in the presence or absence of immunizing peptide, permitting the specifically precipitated proteins to be identified from background non-specifically adsorbed proteins. We confirmed that immobilized v-erb B protein from cell lysates of unlabelled AEV-infected chicken erythroblasts became labelled upon incubation with [gamma-32P]ATP. In addition we demonstrated for the first time that v-erb B from mammalian cells became labelled under the same conditions. These results suggest that the v-erb B protein may possess intrinsic kinase activity. The reagents described should permit further investigations as to whether this activity plays a role in maintaining cellular transformation.

Topics: Adenosine Triphosphate; Alpharetrovirus; Amino Acid Sequence; Animals; Antibodies; Cell Transformation, Viral; Chickens; Erythroblasts; Fibroblasts; Peptides; Phosphorus Radioisotopes; Platelet-Derived Growth Factor; Protein Kinases; Rats; Viral Proteins

Reduction of the survival of motor neurons (SMN) protein levels causes the motor neuron degenerative disease spinal muscular atrophy, the severity of which correlates with the extent of reduction in SMN. SMN, together with Gemins 2 to 7, forms a complex that functions in the assembly of small nuclear ribonucleoprotein particles (snRNPs). Complete depletion of the SMN complex from cell extracts abolishes snRNP assembly, the formation of heptameric Sm cores on snRNAs. However, what effect, if any, reduction of SMN protein levels, as occurs in spinal muscular atrophy patients, has on the capacity of cells to produce snRNPs is not known. To address this, we developed a sensitive and quantitative assay for snRNP assembly, the formation of high-salt- and heparin-resistant stable Sm cores, that is strictly dependent on the SMN complex. We show that the extent of Sm core assembly is directly proportional to the amount of SMN protein in cell extracts. Consistent with this, pulse-labeling experiments demonstrate a significant reduction in the rate of snRNP biogenesis in low-SMN cells. Furthermore, extracts of cells from spinal muscular atrophy patients have a lower capacity for snRNP assembly that corresponds directly to the reduced amount of SMN. Thus, SMN determines the capacity for snRNP biogenesis, and our findings provide evidence for a measurable deficiency in a biochemical activity in cells from patients with spinal muscular atrophy.

Topics: Animals; Biotinylation; Cell Extracts; Cell Line; Cell Line, Transformed; Cell Transformation, Viral; Chickens; Cyclic AMP Response Element-Binding Protein; Cytoplasm; Fibroblasts; HeLa Cells; Herpesvirus 4, Human; Humans; Kinetics; Models, Biological; Motor Neurons; Muscular Atrophy, Spinal; Nerve Tissue Proteins; Phosphorus Radioisotopes; Protein Binding; Ribonucleoproteins, Small Nuclear; RNA-Binding Proteins; RNA, Small Nuclear; Sensitivity and Specificity; SMN Complex Proteins; Transcription, Genetic

The in vivo labelling of viral or cellular components is usually conducted through the addition of radioactively labelled precursors to the culture medium. A limiting factor for isotope use is often the cost of isotope purchase and disposal. Therefore, significant savings can be achieved if the smallest possible volume of medium is employed. However, in the case of adherent cells growing in tissue culture dishes or multi-well plates, surface tension causes a very uneven distribution of the liquid due to the formation of a meniscus at the edge of the petri. This prevents the use of very small volumes of medium for cell growth and labelling because the cells at the center of the petri dish would dry out and die, especially after longer incubation periods. In this communication, we describe a technique whereby cells are grown in an area surrounded by a hydrophobic ring of Teflon, which greatly improves the distribution of the medium by eliminating the concave meniscus. This translates into a dramatic improvement in the condition of the cells, as well as the efficiency of labelling of phosphoproteins, such as the Simian Virus 40 large tumor antigen with 32P-orthophosphate or labelling of the cellular DNA with 3[H]thymidine. The technique is useful for any application where growth of cells in small volumes of medium is required.

Topics: Animals; Antigens, Viral, Tumor; Cell Adhesion; Cell Extracts; Cell Line, Transformed; Cell Transformation, Viral; Culture Media; DNA, Viral; Phosphorus Radioisotopes; Polytetrafluoroethylene; Rats; Silicon; Simian virus 40; Surface Tension; Tritium; Virus Cultivation; Viruses

It has been reported previously that oncogenically transformed cells secrete different molecular forms of osteopontin (OPN), a sialic acid-rich, adhesive, phosphoglycoprotein, than OPNs secreted by their nontransformed counterparts. However, the origin of the OPN isoform secreted by the transformed cells and whether it has different physiological properties which may serve transformation-specific functions remain poorly understood. Here, we report that Rat-1 cells transformed by a temperature-sensitive mutant of Rous sarcoma virus (tsB77) secrete two discrete molecular forms of OPN, a 69-kDa OPN at the nonpermissive temperature (41 degrees C) and a 62-kDa form at the permissive temperature (34 degrees C). However, tsB77 cells at both temperatures transcribe a single 1.6 kb OPN mRNA and contain only the 69-kDa form of OPN intracellularly, suggesting that the 69-kDa OPN is modified to the 62-kDa form prior to or immediately after secretion by cells at 34 degrees C. We ruled out proteolytic cleavage, differential phosphorylation, or lack of N- or O-linked carbohydrates as the possible mechanism, but found that the 62-kDa OPN contains significantly reduced levels of sialic acid, as compared to its 69-kDa form. The binding assays using 32P-labeled OPN revealed that only the 69-kDa OPN, not its 62-kDa form, undergoes receptor-mediated localization on the cell surface, although tsB77 cells synthesize OPN receptors (alpha(v)beta3 integrins) at both permissive and nonpermissive temperatures. Furthermore, 125I-labeled purified milk OPN, which is highly sialylated and shows cell surface binding, upon digestion with neuraminidase failed to interact with the cell surface. Taken together, these results suggest that the difference between the 69-kDa and 62-kDa isoforms of OPN resides in their sialic acid content, and sialylation of OPN is crucial for its receptor-mediated binding on tsB77 cells. The data presented here demonstrate for the first time a physiological role of sialic acids in this protein, and raise the possibility that oncogenically transformed tsB77 cells may exploit the lack of OPN-receptor interactions for their invasive behavior.

Topics: Animals; Avian Sarcoma Viruses; Cell Division; Cell Transformation, Neoplastic; Cell Transformation, Viral; Glycosylation; Humans; Kinetics; Milk Proteins; Molecular Weight; N-Acetylneuraminic Acid; Osteopontin; Peptide Mapping; Phosphates; Phosphopeptides; Phosphorus Radioisotopes; Protein Binding; Rats; Receptors, Cell Surface; Serine Endopeptidases; Sialoglycoproteins; Staphylococcus aureus; Temperature; Trypsin; Tumor Cells, Cultured

An analysis was made of the retroviral integration sites for retroviruses in a murine lymphoid precursor cell line, C1-V13D, derived following in vitro infection with RadLV, an ecotropic murine retrovirus. A genomic library was constructed and lambda clones were selected for their capacity to hybridize with the specific RadLV gp70 ecotropic env probe. Analysis of these clones by a combination of approaches, including subcloning, partial restriction mapping and sequencing, has confirmed the existence of multiple recombinant and defective viruses in C1-V13D. To check for the presence of coding sequences in flanking genomic DNA, 32P-labelled cDNA from C1-V13D was used to probe HindIII- and Psti-digested virus-positive lambda clones by Southern analysis. Regions hybridizing specifically with 32P-labelled C1-V13D cDNA were subcloned and analysed. A notable feature of these cDNA+ regions was the frequent presence of B1, B2 and simple repeats. These repeat elements were found to be present in high frequency in the genomic regions flanking the proviruses, in numbers higher than expected for the genome as a whole. All full-length viruses isolated appeared to represent integration events into regions rich in repeat elements. Some B1 and B2 repeats have been shown to code for functional proteins and to play regulatory roles. Viral integration in the vicinity of these genetic elements could contribute to oncogenesis if the integration event were to disrupt normal gene function.

Topics: Animals; Cell Transformation, Viral; DNA, Neoplasm; DNA, Viral; Genome, Viral; Leukemia, Experimental; Mice; Phosphorus Radioisotopes; Repetitive Sequences, Nucleic Acid; Retroviridae; Retroviridae Proteins, Oncogenic; Tumor Cells, Cultured; Viral Envelope Proteins; Virus Integration

Immobilon, a membrane of polyvinylidene difluoride to which gel-fractionated proteins can be transferred electrophoretically, was found to be an excellent matrix for the analysis of the phosphoamino acid content of phosphoproteins. Hydrolysis of 32P-labeled proteins bound to Immobilon with 5.7 N HCl resulted in the release of 90% of the 32P in the form of Pi, phosphoamino acids, and phosphopeptides. Two-dimensional electrophoretic analysis of the released phosphoamino acids yielded undistorted patterns. Because direct hydrolysis of proteins transferred to Immobilon eliminated the need for both preparative extraction of proteins from a gel and recovery by precipitation, analysis was rapid and yields of phosphoamino acids were extremely consistent. The yield of phosphoamino acids from proteins bound to Immobilon, unlike that from proteins eluted from gels, was independent of the size of the protein. The detection of 32P-labeled, phosphotyrosine-containing proteins in sodium dodecyl sulfate-polyacrylamide gels has been shown to be substantially improved by incubation of the gel in 1.0 N KOH for 2 h at 55 degrees C. Base hydrolysis of proteins bound to Immobilon proved to be faster and more sensitive than hydrolysis of proteins in gels. Less than 10% of bound protein was lost from Immobilon during the 2-h incubation at 55 degrees C in 1.0 N KOH. The autoradiographic image after alkaline hydrolysis of proteins on Immobilon was sharper than that obtained after hydrolysis of proteins in the gel. In addition, unlike base-treated gels, the dimensions of the Immobilon filter were unaffected by treatment with base.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acids; Animals; Avian Sarcoma Viruses; Cell Transformation, Viral; Cells, Cultured; Chromatography, Affinity; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Hydrolysis; Indicators and Reagents; Mice; Mice, Inbred BALB C; Phosphoproteins; Phosphorus Radioisotopes; Polyvinyls; Radioisotope Dilution Technique

We have developed and compared two DNA dot hybridization methodologies with similar probes (radioisotope-labelled and biotin-labelled) to detect varicella-zoster virus (VZV) DNA in three different cell cultures at varying times post-infection. Control cultures included uninfected monolayers of the same cells. Cellular DNA was isolated by a standard phenol extraction method, after which the DNA was quantified, serially diluted and blotted onto nitrocellulose or nylon membranes. The VZV DNA probe, which consisted of the large Hind III A fragment (27 of the total 125 kbp), was produced in two separate nick translation systems. The first contained 100 microCi [32P] and 0.4 microgram Hind III fragment A of the varicella genome, while the second probe employed a biotin-7-dATP analogue and 1.0 microgram of the Hind III fragment A. Direct visualization on the membrane or the exposed radiographic film showed a dot of varying intensity whenever viral genome was detected with either the biotin or the radioactive probe, respectively. With the [32P]-labelled probe, we detected VZV genomic sequences within 0.5 microgram total DNA at 12 h post-infection. This amount corresponded to approximately 5-10 pg of viral DNA. By comparison, hybridization with the biotin-labelled probe required 0.5-1.0 micrograms total DNA from infected cells. Similar tests on DNA extracted from uninfected cell samples were negative with both probes.

Topics: Biotin; Cell Line; Cell Transformation, Viral; Cells, Cultured; Cornea; DNA Probes; DNA, Viral; Genes, Viral; Herpesvirus 3, Human; Humans; Melanoma; Nucleic Acid Hybridization; Phosphorus Radioisotopes; Protein Biosynthesis

Methods have been developed and applied to determine the size and branching frequency of polymers of ADP-ribose synthesized in nucleotide-permeable cultured mouse cells and in intact cultured cells. Polymers were purified by affinity chromatography with a boronate resin and were fractionated according to size molecular sieve high-performance liquid chromatography. Fractions were enzymatically digested to nucleotides, which were separated by strong anion exchange high-performance liquid chromatography. From these data, average polymer size and branching frequency were calculated. A wide range of polymer sizes was observed. Polymers as large as 190 residues with at least five points of branching per molecule were generated in vitro. Polymers of up to 67 residues containing up to two points of branching per molecule were isolated from intact cells following treatment with the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. Cells treated with hyperthermia prior to DNA damage contained polymers of an average maximum size of 244 residues containing up to six points of branching per molecule. The detection of large polymers of ADP-ribose in intact cells suggests that alterations in chromatin organization effected by poly(ADP-ribosylation) may extend beyond the covalently modified proteins and very likely involve noncovalent interactions of poly(ADP-ribose) with other components of chromatin.

Topics: Animals; Cell Membrane Permeability; Cell Transformation, Viral; Cells, Cultured; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Hot Temperature; Kinetics; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; NAD; Nucleoside Diphosphate Sugars; Phosphorus Radioisotopes; Poly Adenosine Diphosphate Ribose; Simian virus 40

The proteins associated with parental, adenoviral DNA in productively-infected HeLa cells have been examined both directly and indirectly. HeLa cells infected with 32P-labelled Ad2 were irradiated with u.v. light at various points in the infectious cycle. Following degradation of the DNA, nuclear proteins carrying cross-linked nucleotides, or oligonucleotides, were distinguished from virion phosphoproteins by the resistance of their 32P radioactivity to 1 M NaOH. The major core protein of the virion, protein VII, was found to be associated with viral DNA throughout infection, even when cells were infected at a multiplicity of 0.14. Micrococcal nuclease digestion of intranuclear viral DNA 4 h after infection liberated two nucleoprotein particles containing viral DNA, neither of which co-migrated with HeLa cell mononucleosomes. These results indicate that core protein VII remains associated with parental adenoviral DNA during productive infections. The observation that protein VII can be cross-linked to DNA in cells infected at very low multiplicity, together with the results of a comparison of proteins cross-linkable to viral DNA in cells infected by wild-type virus and a non-infectious mutant containing the precursor to protein VII, suggest that nucleoproteins comprising viral DNA and protein VII must be the templates for expression of pre-early and early viral genes.

Topics: Adenoviruses, Human; Cell Transformation, Viral; DNA, Viral; HeLa Cells; Humans; Kinetics; Phosphoproteins; Phosphorus Radioisotopes; Ultraviolet Rays; Viral Core Proteins; Virion

The consequences of herpes simplex virus type 1 infection on cellular macromolecules were investigated in Friend erythroleukemia cells. The patterns of protein synthesis, examined by polyacrylamide gel electrophoresis, demonstrated that by 4 h postinfection the synthesis of many host proteins, with the exception of histones, was inhibited. Examination of the steady-state level of histone H3 mRNA by molecular hybridization of total RNA to a cloned mouse histone H3 complementary DNA probe demonstrated that the ratio of histone H3 mRNA to total RNA remained unchanged for the first 4 h postinfection. In contrast, the steady-state levels of globin and actin mRNAs decreased progressively at early intervals postinfection. Studies on RNA synthesis in isolated nuclei demonstrated that the transcription of the histone H3 gene was inhibited to approximately the same extent as that of actin gene. We concluded that the stabilization of preexisting histone H3 mRNA was responsible for the persistence of H3 mRNA and histone protein synthesis in herpes simplex virus type 1-infected Friend erythroleukemia cells. The possible mechanisms influencing the differential stability of host mRNAs during the course of productive infection with herpes simplex virus type 1 are discussed.

Topics: Animals; Cell Line; Cell Transformation, Viral; DNA; DNA Replication; Histones; Kinetics; Leukemia, Experimental; Mice; Nucleic Acid Hybridization; Phosphorus Radioisotopes; Protein Biosynthesis; RNA, Messenger; Simplexvirus; Transcription, Genetic; Uridine Triphosphate

The association of adenoviral DNA with the high salt (2 M NaCl) resistant nuclear fraction, termed the nuclear matrix, has been investigated in HeLa cells at different times after infection with adenovirus type 5. When nuclear matrices were prepared in the absence of exogenously added nucleases, Ad5 DNA was quantitively associated with the matrix throughout the infection period (0-24 h). Moreover, early in infection (0-10 h) Ad5 DNA was severalfold enriched in DNase I digested nuclear matrices (8-15% of total nuclear DNA) compared to the high salt soluble chromatin fraction (85-90% of total nuclear DNA). At later times after infection, progressively more Ad5 DNA appeared in the chromatin fraction until, at 24 h, the nuclear matrix was strikingly depleted in Ad5 DNA. A large proportion of the Ad5 DNA in nuclear matrices prepared early in infection, e.g., 4 h, was full length in size. At later times (12-24 h) most of the viral DNA was fragmented to a size equivalent to total matrix DNA (100-1000 base pairs). The apparent switch of the matrix-associated viral DNA from a relatively DNase I resistant to sensitive state was initiated approximately at the time when viral DNA replication began (12 h). Since no discrete portion of the Ad5 genome was significantly enriched at the sites of attachment to the nuclear matrix throughout the infection period, the switch in DNase I sensitivity is not mediated by a change in DNA sequence attachment to the matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenoviruses, Human; Cell Nucleus; Cell Transformation, Viral; Deoxyribonuclease I; DNA, Viral; HeLa Cells; Humans; Kinetics; Nucleic Acid Hybridization; Phosphorus Radioisotopes

Runoff transcripts were generated on viral transcriptional complexes cleaved with restriction enzymes and incubated in vitro with [alpha-32P]UTP under pulse-chase conditions. As viral transcriptional complexes in vitro elongated the nascent RNA preinitiated in vivo, size analysis by gel electrophoresis of the runoff transcripts allowed identification of the in vivo initiation sites. Moreover, scanning the intensities of the runoff bands as they appeared in the autoradiogram of the gel allowed determination of the relative use of these sites. A model system in which the initiation sites of simian virus 40 late RNA were identified and their relative use determined is presented.

Topics: Animals; Base Sequence; Cell Line; Cell Transformation, Viral; Chlorocebus aethiops; DNA Restriction Enzymes; Kidney; Phosphorus Radioisotopes; RNA, Viral; Simian virus 40; Transcription, Genetic; Uridine Triphosphate

Tyrosyl kinase activity in vesicular stomatitis virus (VSV) acquired from host cells that differ in morphology was investigated. VSV grown in baby hamster kidney (BHK) cells with rounded morphology and a high efficiency of colony formation in soft agar (Rous sarcoma virus [RSV]-transformed and suspension BHK cells) was compared with VSV grown in BHK cells with a flattened morphology and lower efficiency of colony formation in soft agar (RSV-infected revertant and control BHK cells). Tyrosyl kinase activity measured with the substrates angiotensin II peptide or casein was found at 7-10-fold higher levels in virus released from the anchorage-independent BHK cells. Most of the VSV-associated tyrosyl kinases acquired from the RSV-transformed BHK cells reacted with antiserum to pp60src, whereas the activity acquired from the suspension BHK cells was unaffected by anti-src serum. The overall levels of tyrosyl kinase in subcellular fractions of the host BHK cells were also measured. Like the VSV released from them, the RSV-transformed cell extracts contained high levels. The suspension cells, however, contained the same low levels of tyrosyl kinase as was found in the control BHK cell extracts. Therefore, tyrosyl kinase was concentrated and acquired by VSV from the anchorage-independent suspension BHK cells. VSV-associated protein kinases acquired from other cell types followed a similar pattern. Tyrosyl kinase levels were high in VSV released from suspension cultures (Chinese hamster ovary and HeLa) and from virally transformed cells (Kirsten murine sarcoma virus-transformed rat kidney cells) and low in VSV released from an anchorage-dependent primary cell culture (chick embryo fibroblasts).

Topics: Amino Acids; Animals; Cell Adhesion; Cell Line; Cell Transformation, Viral; Cricetinae; Kidney; Kinetics; Phosphorus Radioisotopes; Phosphotyrosine; Protein Kinases; Protein-Tyrosine Kinases; Tyrosine; Vesicular stomatitis Indiana virus; Virion

A technique which detects viral DNA or RNA in situ in the organ systems of whole mice is described. Frozen thin sections from whole mice were transferred directly to nitrocellulose and hybridized to labeled viral DNA, allowing the detection of viral DNA or RNA. By this procedure, polyomavirus infection of newborn mice inoculated intranasally was followed. We found that the initial inoculum could be detected in the nasal cavity, lungs, and stomach lining after a 5-h absorption period. Primary replication of virus was observed in the nasal cavity, submaxillary gland, and lungs, followed by a systemic phase of infection in which the liver, spleen, kidney, and large colon also became infected. Viral RNA as well as DNA could also be detected as shown by infecting mice intracerebrally with vesicular stomatitis virus. Vesicular stomatitis virus-specific RNA was observed only in the brains of these mice. It is most likely that this technique can be applied to general molecular studies of mice. With this method we should be able to detect all viruses, bacteria, plasmids, and organ-specific transcripts to which a cloned probe exists.

Topics: Animals; Animals, Newborn; Autoradiography; Cell Transformation, Viral; DNA, Viral; Genes, Viral; Mice; Mice, Inbred BALB C; Nucleic Acid Hybridization; Organ Specificity; Phosphorus Radioisotopes; Polyomavirus; RNA, Viral; Spleen

A new molecular hybridization approach to the analysis of complex genomes has been developed. Tracer and driver DNAs were digested with the same restriction enzyme(s), and tracer DNA was labeled with 32P using T4 DNA polymerase. Tracer DNA was mixed with an excess amount of driver, and the mixture was electrophoresed in an agarose gel. Following electrophoresis, DNA was alkali-denatured in situ and allowed to reanneal in the gel, so that tracer DNA fragments could hybridize to the driver only when homologous driver DNA sequences were present at the same place in the gel, i.e. within a restriction fragment of the same size. After reannealing, unhybridized single-stranded DNA was digested in situ with S1 nuclease. The hybridized tracer DNA was detected by autoradiography. The general applicability of this technique was demonstrated in the following experiments. The common EcoRI restriction fragments were identified in the genomes of E. coli and four other species of bacteria. Two of these fragments are conserved in all Enterobacteriaceae. In other experiments, repeated EcoRI fragments of eukaryotic DNA were visualized as bands of various intensity after reassociation of a total genomic restriction digest in the gel. The situation of gene amplification was modeled by the addition of varying amounts of lambda phage DNA to eukaryotic DNA prior to restriction enzyme digestion. Restriction fragments of lambda DNA were detectable at a ratio of 15 copies per chicken genome and 30 copies per human genome. This approach was used to detect amplified DNA fragments in methotrexate (MTX)-resistant mouse cells and to identify commonly amplified fragments in two independently derived MTX-resistant lines.

Topics: Cell Line; Cell Transformation, Viral; DNA; DNA Restriction Enzymes; DNA-Directed DNA Polymerase; Enterobacteriaceae; Escherichia coli; Gene Amplification; Genes, Bacterial; Herpesvirus 4, Human; Humans; Lymphocytes; Nucleic Acid Hybridization; Nucleic Acid Renaturation; Phosphorus Radioisotopes; T-Phages

Topics: Animals; Cell Transformation, Viral; Cells, Cultured; Kinetics; Mice; Phosphorus Radioisotopes; Phosphorylation; Radioisotope Dilution Technique; Ribosomal Protein S6; Ribosomal Proteins; Simian virus 40

Pyridoxal [32P] phosphate was prepared using [gamma-32P] ATP, pyridoxal, and pyridoxine kinase purified from Escherichia coli B. The pyridoxal [32P] phosphate obtained had a specific activity of at least 1 Ci/mmol. This reagent was used to label intact influenza virus, red blood cells, and both normal and transformed chick embryo fibroblasts. The cell or virus to be labeled was incubated with pyridoxal [32P] phosphate. The Schiff base formed between pyridoxal [32P] phosphate and protein amino groups was reduced with NaBH4. The distribution of pyridoxal [32P] phosphate in cell membrane or virus envelope proteins was visualized by autoradiography of the proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The labeling of the proteins of both influenza and chick cells appeared to be limited exclusively to those on the external surface of the virus or plasma membrane. With intact red blood cells the major portion of the probe was bound by external proteins, but a small amount of label was found associated with the internal proteins spectrin and hemoglobin.

Topics: Avian Sarcoma Viruses; Cell Transformation, Neoplastic; Cell Transformation, Viral; Erythrocyte Membrane; Erythrocytes; Escherichia coli; Fibroblasts; Membrane Proteins; Neoplasm Proteins; Orthomyxoviridae; Phosphorus Radioisotopes; Phosphotransferases; Pyridoxal Phosphate; Pyridoxine; Schiff Bases; Viral Proteins