Biomedical subjects

R H Singer

Publications and source records attributed to R H Singer.

At least 37 records · Page 2Linked to original sources

PubMed1989

Temporal resolution and sequential expression of muscle-specific genes revealed by in situ hybridization.

The expression of muscle-specific mRNAs was analyzed directly within individual cells by in situ hybridization to chicken skeletal myoblasts undergoing differentiation in vitro. The probes detected mRNAs for sarcomeric myosin heavy chain (MHC) or the skeletal, cardiac, and beta isoforms of actin. Precise information as to the expression of these genes in individual cells was obtained and correlated directly with analyses of cell morphology and interactions, cell cycle stage, and immunofluorescence detection of the corresponding proteins. Results demonstrate that mRNAs for the two major muscle-specific proteins, myosin and actin, are not synchronously activated at the time of cell fusion. The mRNA for alpha-cardiac actin (CAct), known to be the predominant embryonic actin isoform in muscle, is expressed prior to cell fusion and prior to the expression of any isoform of muscle MHC mRNA. MHC mRNA accumulates rapidly immediately after fusion, whereas skeletal actin mRNA is expressed only in larger myofibers. Single cells expressing CAct mRNA have a characteristic short bipolar morphology, are in terminal G1, and do not contain detectable levels of the corresponding protein. In a pattern of expression reciprocal to that of CAct mRNA, beta-actin mRNA diminishes to low or undetectable levels in myofibers and in cells of the morphotype which expresses CAct mRNA. Finally, the intracellular distribution of mRNAs for different actin isoforms was compared using nonisotopic detection of isoform-specific oligonucleotide probes. This work illustrates a generally valuable approach to the analysis of cell differentiation and gene expression which directly integrates molecular, morphological, biochemical, and cell cycle information on individual cells.

Actins↗

PubMed1989

Ultrastructural visualization of cytoskeletal mRNAs and their associated proteins using double-label in situ hybridization.

We have been able to visualize cytoskeletal messenger RNA molecules at high resolution using nonisotopic in situ hybridization followed by whole-mount electron microscopy. Biotinated cDNA probes for actin, tubulin, or vimentin mRNAs were hybridized to Triton-extracted chicken embryo fibroblasts and myoblasts. The cells were then exposed to antibodies against biotin followed by colloidal gold-conjugated antibodies and then critical-point dried. Identification of mRNA was possible using a probe fragmented to small sizes such that hybridization of several probe fragments along the mRNA was detected as a string of colloidal gold particles qualitatively and quantitatively distinguishable from nonspecific background. Extensive analysis showed that when eight gold particles were seen in this iterated array, the signal to noise ratio was greater than 30:1. Furthermore, these gold particles were colinear, often spiral, or circular suggesting detection of a single nucleic acid molecule. Antibodies against actin, vimentin, or tubulin proteins were used after in situ hybridization, allowing simultaneous detection of the protein and its cognate message on the same sample. This revealed that cytoskeletal mRNAs are likely to be extremely close to actin protein (5 nm or less) and unlikely to be within 20 nm of vimentin or tubulin filaments. Actin mRNA was found to be more predominant in lamellipodia of motile cells, confirming previous results. These results indicate that this high resolution in situ hybridization approach is a powerful tool by which to investigate the association of mRNA with the cytoskeleton.

Actins↗

PubMed1989

Detection of Epstein-Barr virus by in situ hybridization. Progress toward development of a nonisotopic diagnostic test.

This work presents some initial quantitation of an in situ hybridization method for detection of Epstein-Barr (EB) virus nucleic acids. The purpose is to develop evaluative criteria for diagnosis of viral presence in clinical tissue specimens. In this work simultaneous denaturation of probe and target DNA and an alkaline phosphatase conjugate to detect biotinated probe were used as described by Unger et al. For evaluation of the hybridization, a variety of cell lines, both productively and latently infected, that were hybridized in situ using nick translated 32P-labeled viral probe sequences and counted by scintillation after the method of Lawrence and Singer were used. Producer cells (B95-8) showed intense foci of staining in approximately 5% of cells, with most of the other cells showing varying staining intensity. Raji cells showed varying amounts of signal from cell to cell. Namalwa cells exhibited one spot in most cells that was decreased after cells were treated with Actinomycin D (dactinomycin, Merck Sharp & Dohme, West Point, PA). Signal was identified in only a third of these same cells after sectioning. EB virus-negative Ramos cells showed no signal. The nuclear punctate nature of the signal generated is diagnostic of infected cells, and may be a useful test for cultured cells or pathologic specimens.

Autoradiography↗

PubMed1988

Sensitive, high-resolution chromatin and chromosome mapping in situ: presence and orientation of two closely integrated copies of EBV in a lymphoma line.

Here we describe development and application of highly sensitive fluorescence methodology for localization of single-copy sequences in interphase nuclei and metaphase chromosomes by nonisotopic in situ hybridization. Application of this methodology to the investigation of Epstein-Barr virus integration in the Namalwa lymphoma cell line has revealed two EBV genomes closely integrated at the known site on chromosome 1. Detecting sequences as small as 5 kb, we further demonstrate resolution within interphase nuclei of two fragments of the viral genome spaced only 130 kb apart. Results indicate that the viral genomes are in opposite orientations and separated by roughly 340 kb of cellular DNA. This work demonstrates the feasibility and resolving power of interphase chromatin mapping to assess the proximity of closely spaced DNA sequences. Implications for virology, gene mapping, and investigation of nuclear organization are discussed.

Base Sequence↗

PubMed1988

Intracellular distribution of histone mRNAs in human fibroblasts studied by in situ hybridization.

We have used in situ hybridization to study the intracellular distribution of mRNAs for cell cycle-dependent core and H1 histone proteins in human WI-38 fibroblasts. Because histones are abundant nuclear proteins and histone mRNA expression is tightly coupled to DNA synthesis, it was of interest to determine whether histone mRNAs are localized near the nucleus. Cells were hybridized with tritiated DNA probes specific for either histone H1, histone H4, actin, or poly(A)+ mRNA and were processed for autoradiography. In exponentially growing cultures, the fraction of histone mRNA-positive cells correlated well with the fraction of cells in S phase and was eliminated by hydroxyurea inhibition of DNA synthesis. Within individual cells the label for histone mRNA was widely distributed throughout the cytoplasm and did not appear to be more heavily concentrated near the nucleus. However, histone mRNA appeared to exhibit patchy, nonhomogeneous localization, and a quantitative evaluation confirmed that grain distributions were not as uniform as they were after hybridizations to poly(A)+ mRNA. Actin mRNA in WI-38 cells was also widely distributed throughout the cytoplasm but differed from histone mRNA in that label for actin mRNA was frequently most dense at the outermost region of narrow cell extensions. The localization of actin mRNA was less pronounced but qualitatively very similar to that previously described for chicken embryonic myoblasts and fibroblasts. We conclude that localization of histones in WI-38 cells is not facilitated by localization of histone protein synthesis near the nucleus and that there are subtle but discrete and potentially functional differences in the distributions of histone, actin, and poly(A)+ mRNAs.

Actins↗

PubMed1987

Use of oligodeoxynucleotide probes for quantitative in situ hybridization to actin mRNA.

We have employed an analytical approach for the development of an in situ hybridization methodology using synthetic oligodeoxynucleotide probes for actin messenger RNA detection in cultures of chicken fibroblasts and myoblasts. The methodology developed shows that oligonucleotides can complement the use of nick-translated probes in specific situations. Since they can be made to specific nucleic acid regions independent of restriction enzyme sites, they may be the most convenient approach for analysis of gene families among which sequences are highly conserved. However, it was found that oligonucleotides synthesized to different regions of a messenger RNA behave in situ with differing efficiencies, indicating that not all target sequences are equivalent. Therefore it was necessary to screen several oligonucleotide probes to a target molecule to find the optimal one. The convenience of using synthetic DNA probes makes it worthwhile to explore some of these characteristic properties so as to increase the sensitivity of this approach beyond its application to targets in high abundance.

Actins↗

PubMed1987

Amino acid sequences that determine the nuclear localization of yeast histone 2B.

Histone-beta-galactosidase protein fusions were used to identify the domain of yeast histone 2B, which targets this protein to the nucleus. Amino acids 28 to 33 in H2B were required for nuclear localization of such fusion proteins and thus constitute a nuclear localization sequence. The amino acid sequence in this region (Gly-29 Lys Lys Arg Ser Lys Ala) is similar to the nuclear location signal in simian virus 40 large T antigen (Pro-126 Lys Lys Lys Arg Lys Val) (D. Kalderon, B.L. Roberts, W.D. Richardson, and A.E. Smith, Cell 39:499-509, 1984). A point mutation changing lysine 31 to methionine abolished nuclear localization of an H2B-beta-galactosidase fusion protein containing amino acids 1 to 33 of H2B. However, an H2B-beta-galactosidase fusion protein containing both this point mutation and the H2A interaction domain of H2B was nuclear localized. These results suggest that H2A and H2B may be cotransported to the nucleus as a heterodimer.

Amino Acid Sequence↗

PubMed1986

Intracellular localization of messenger RNAs for cytoskeletal proteins.

We have analyzed intracellular distributions of mRNAs for the cytoskeletal proteins actin, vimentin, and tubulin by in situ hybridization. Although polyadenylated RNA was homogeneously distributed throughout the cell, actin mRNA demonstrated a nonhomogeneous distribution in 95% of randomly selected chicken embryonic myoblasts and fibroblasts, as detected by isotopic and nonisotopic techniques. Actin mRNA concentrations were highest at cell extremities, generally in lamellipodia, where grain densities were up to 16-fold higher than in areas near the nucleus. Vimentin mRNA, unlike actin mRNA, was distributed near the nucleus. Tubulin mRNA appeared most concentrated in the peripheral cytoplasm. These results demonstrate that cytoplasmic mRNAs are localized in specific, nonrandom cellular patterns and that localized concentrations of specific proteins may result from corresponding localization of their respective mRNAs. Hence, actin mRNA distribution may result in increased concentration of actin filaments in lamellipodia of motile cells.

Actins↗

PubMed1986

Location of a polypeptide sequence within the alpha-subunit of the acetylcholine receptor containing the cholinergic binding site.

Proteolytic fragments of the alpha-subunit of the acetylcholine receptor of Torpedo electric organ were generated by digestion with Staphylococcus aureus V8 protease, and their ability to bind alpha-bungarotoxin was assessed following resolution on polyacrylamide gels and transfer to nitrocellulose. The position of the smallest fragment (Mr = 17,000) with toxin-binding activity was located within the primary sequence of the alpha-subunit by isolation and chemical characterization. The amino acid sequence at its amino terminus is Val-Asn-Gln-Ile-Val-Glu, which is identical to a unique sequence on the alpha-subunit beginning at Val 46. Based on considerations of the apparent molecular weight of this polypeptide fragment, the enzyme specificity of V8 protease, analysis of the partial amino acid composition, and the position of various identifying landmarks of the primary sequence of the alpha-subunit, the carboxy terminus is restricted to an acidic amino acid residue between Asp 152 and Asp 180. The apparent affinities of the 17-kDa polypeptide for alpha-bungarotoxin (IC50 = 100 nM) and d-tubocurarine (IC50 = 0.4 mM) were not significantly different from the values obtained for the undigested alpha-subunit, suggesting that the 17-kDa polypeptide contains all of the essential elements present on the alpha-subunit that are necessary for the binding of both of these cholinergic ligands. A second toxin-binding fragment (Mr = 19,000) was also identified. Although the amino terminus of this polypeptide fragment has not been determined directly, its position relative to Asn 141, the site of N-glycosylation, was established, and it appears that its amino terminus cannot be located any closer to the amino terminus of the alpha-subunit than Asp 152. If this tentative assignment is correct, then the region of the alpha-subunit involved in binding cholinergic ligands is more closely defined as occurring within the segment of the primary sequence of the alpha-subunit that the 17- and 19-kDa polypeptide fragments have in common, i.e., an overlapping sequence maximally bounded by Asp 152 and Asp 180.

Acetylcholine↗

PubMed1985

Quantitative analysis of in situ hybridization methods for the detection of actin gene expression.

We have implemented an efficient, quantitative approach for the optimization of in situ hybridization using double-stranded recombinant DNA probes. The model system studied was actin mRNA expression in chicken embryonic muscle cultures. Actin and control (pBR322) probes were nick-translated with p32 labeled nucleotides, hybridized to cells grown on coverslips, and quantitated in a scintillation counter. Cellular RNA retention was monitored via the incorporation of H3-Uridine into RNA prior to cell fixation. Over a thousand samples were analyzed, and among the technical variables examined were the fixation protocol, proteolytic cell pretreatment, the time course of hybridization, saturation kinetics, hybridization efficiency, and effect of probe size on hybridization and network formation. Results have allowed us to develop a reproducible in situ hybridization methodology which is simpler and less destructive to cellular RNA and morphology than other protocols. Moreover, this technique is highly sensitive and efficient in detection of cellular RNAs. Lastly, the rapid quantitative approach used for this analysis is valuable in itself as a potential alternative to filter or solution hybridizations.

Actins↗

PubMed1984

Filament-directed intercellular contacts during differentiation of cultured chick myoblasts.

Detergent-extracted, critical point dried chicken myoblasts at early stages of development in tissue culture were observed by electron microscopy. It was found that the organization of filaments within these cells changes significantly during development. A particular specialization of the cellular filament framework is the formation of microprocesses; long extensions of the cellular filament system. These microprocesses appear to be involved in cell-to-cell contact. The filaments of these processes appear to integrate with the filament system of a contacted cell, and possibly transmit tension from one cell to another. The role of these structures in effecting muscle differentiation by forming cytoplasmic connections and the implications for muscle gene expression are discussed.

Animals↗

PubMed1983

Analysis of receptor-ligand interactions using nitrocellulose gel transfer: application to Torpedo acetylcholine receptor and alpha-bungarotoxin.

A nitrocellulose-gel transfer technique has been adapted to study the interaction of a polypeptide ligand with individual receptor subunits. The acetylcholine receptor isolated from Torpedo californica has been separated into its subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred in a renaturing environment to nitrocellulose sheets. The sheets were incubated with 125I-alpha-bungarotoxin and autoradiographed. A single receptor polypeptide, the alpha subunit (40K) bound the labeled toxin. This binding was demonstrated to be both saturable and specific, although the affinity of 125I-alpha-bungarotoxin (KD, 165 nM) and the potency of d-tubocurarine to displace this binding (IC50, 1 mM) were both reduced by several orders of magnitude when compared to the native receptor.

Animals↗

PubMed1982

Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog.

The chicken muscle tissue culture system has been used for visualizing actin gene expression after in situ hybridization. Cell differentiation is morphologically distinguishable in this system as the myoblasts fuse into myotubes. This differentiation involves the production of large amounts of actin required for myofibrils. The presence of actin mRNA has been observed in cells preserved with ethanol and paraformaldehyde by hybridizing a recombinant plasmid into which a biotinated analog of dUTP was incorporated by nick-translation. The biotin was then detected by using an anti-biotin antibody and a rhodamine-conjugated second antibody. Alternatively, avidin conjugated to rhodamine or avidin complexed to biotinated peroxidase has been used for mRNA detection. The procedure described preserved morphological detail yet is compatible with hybridization conditions and reveals the disposition of actin mRNA during gene expression.

Actins↗

PubMed1981

Increased turnover of proteins from the sarcoplasmic reticulum of dystrophic chicken muscle cells in tissue culture.

Chicken myoblasts were cultured from the pectoralis muscles of dystrophic and normal 11-day-old embryos. Cells were allowed to grow to fusion (differentiation) and exposed to [35S]methionine for a short period. Subsequently, the decay of labeled proteins in the presence of cycloheximide was measured for various cellular fractions as well as individual proteins isolated from the sarcoplasmic reticulum and separated by gel electrophoresis. Some dystrophic material showed an increased decay when compared to normal material. The most significant (p less than 0.005) difference was found in a Mr = 65,000 component of the sarcoplasmic reticulum. This same component accumulates label at an accelerated rate in the presence of the protease inhibitor leupeptin. Increased turnover of this protein, possibly calsequestrin, may be a manifestation of the genetic disease.

Animals↗

PubMed1981

Sea urchin tube feet: unique structures that allow a cytological and molecular approach to the study of actin and its gene expression.

Actin is the major extractable protein component from the tube feet of four different species of sea urchin: Arbacia punctulata, Strongylocentrotus purpuratus, Strongylocentrotus droebachiensis, and Diadema setosum. Actin made up as much as 60% of the total Coomassie Blue-staining material after SDS polyacrylamide gel electrophoresis and densitometer analysis. Two-dimensional gel electrophoresis resolved two, and possible three, species of actin for each sea urchin of which the dominant component was analogous to the beta form in vertebrates. In a cell-free system from rabbit reticulocytes, total RNA from tube feet stimulated the synthesis of one protein that represented 80% of the total methionine incorporation, migrated with the properties characteristic of actin in a two-dimensional gel system, and on proteolysis yielded fragments identical to purified rabbit actin. The mRNAs from the tube feet of two divergent species of sea urchin, Arbacia punctulata and Strongylocentrotus purpuratus, synthesized actins differing by less than 0.02 pH unit for each isospecies 90% of the DNA copied from tube foot RNA by reverse transcriptase represented a highly abundant sequence class judged by copy DNA(cDNA)-RNA excess hybridization. At least two-thirds of this class represented a low-complexity component, with a Rot1/2 about three times that expected for actin messenger RNA. The remarkable degree of conservation of the actin protein is reflected in concomitant conservation of the protein-coding nucleotide sequences of the messenger RNA, which has allowed the use of a cDNA probe to isolate actin sequences from a human phage library.

Actins↗

PubMed1980

Intracellular filament bundles in whole mounts of chick and human myoblasts extracted with triton X-100.

The method of Triton X-100 extraction and critical point drying of whole mounts of cultured chick and human myoblasts was used to study the presence of intracellular bundles of filaments within these cells. Observation by means of transmission and scanning electron microscopy demonstrated a complex system of filament bundles which appeared morphologically and spatially heterogeneous. Most obvious were long dense bundles of cables traversing along the ventral surface of developing myoblasts, presumably the 'stress fibers' seen in light microscopy. Other bundle types occurred which were composed of loose aggregates of filaments coursing through the remnant cell body. A prominent accumulation of filaments was also seen at the lateral edges of these myoblasts. These lateral edge cables were thicker and denser than any other type of filament bundle observed in the myoblasts. Reaction of unextracted myoblasts directly to human antiplatelet myosin conjugated to rhodamine demonstrated that the most intense reaction also occurred along the lateral edges of both human and chick myoblasts. During development of chick myoblasts the filament bundles became oriented parallel to the cell axis giving the cell a fusiform morphology. It is possible that the various filament bundle structures and their differing structural and spatial dispositions could be related to functional differences among the diverse population of intracellular bundles of filaments.

Animals↗

PubMed1979

Electron microscopic visualization of the filamentous reticulum in whole cultured presumptive chick myoblasts.

This present study describes an experimental approach whereby subcellular 3-dimensional filamentous structures present within whole cells can be examined, using a conventional transmission electron microscope. This procedure uses cells which have been cultured on carbon-coated titanium grids, and treated with Triton X-100 to extract the soluble cytoplasm. Subsequent fixation and critical-point drying allows filamentous proteins to be easily visualized, due to the increase in contrast produced by removal of the ground cytoplasm. The high resolution obtainable in these preparations permitted an initial classification and description of the filamentous reticulum within cultured presumptive myoblasts. This reticulum is a continuum of filaments and cables, all elements of which appear to be interconnected. These morphological findings were then correlated with the biochemical identification of detergent-insoluble proteins, of which only actin, myosin, and, perhaps, intermediate filament and LETS protein are the major elements.

Animals↗

PubMed1978

Ca2+ binding, ATP-dependent Ca2+ transport, and total tissue Ca2+ in embryonic and adult avian dystrophic pectoralis.

Avian muscular dystrophy is an autosomal recessive genetic disease characterized by early hypertrophy and loss of function of pectoralis major. The disease is progressive, ultimately resulting in atrophy and heavy lipid deposition. Previous investigators have noted a decrease in the ability of the dystrophic sarcoplasmic reticulum to concentrate Ca2+. More recently, other investigators have shown an abnormal calcium uptake in avian dystrophic sarcoplasmic reticulum. They indicated, using freeze-fracture techniques, that a 90 A particle of the vesicle membrane exhibited a decreased population and suggested that they might be the ATPase involved in calcium transport. Our studies confirm earlier observations of a decreased rate of Ca2+ uptake and Ca2+ binding capacity of dystrophic fragmented sarcoplasmic reticulum vesicles which are isolated from both embryonic and adult pectoralis. These observations correlate in turn with a 75% drop in the Ca:ATP transport efficiency of the dystrophic sarcoplasmic reticulum determined by measuring the rate of 32Pi liberation from gamma-ATP32 during active calcium transport by the isolated sarcoplasmic reticulum SR. In addition, we have found a quantitative deficiency in a 65,000 dalton component of the dystrophic fragmented SR at the time of myoblast fusion by measuring 35S-Methionine incorporation into the SR, coupled to high resolution polyacrylamide gel electrophoresis and radioautography. Analysis of total tissue calcium by atomic absorption spectroscopy revealed a decrease in the total calcium content of dystrophic muscle.

Adenosine Triphosphate↗