Showing papers in "Proceedings of the National Academy of Sciences of the United States of America in 1969"

SUPERCOILED CIRCULAR DNA-PROTEIN COMPLEX IN Escherichia coli: PURIFICATION AND INDUCED CONVERSION TO AN OPEN CIRCULAR DNA FORM

Abstract: The 23S twisted circular form of ColE1 DNA has been isolated from Escherichia coli as a tightly associated DNA-protein complex with a sedimentation coefficient of approximately 24S. Treatment of this complex with pronase, trypsin, sodium dodecyl sulfate, Sarkosyl, or heat results in a conversion to a slower sedimenting form of 17S or 18S, as determined by centrifugation in neutral sucrose gradients. These treatments do not alter the sedimentation properties of noncomplexes supercoiled ColE1 DNA even in the presence of the ColE1-protein complex. Electron microscopic analyses indicate that the decrease in sedimentation rate of the ColE1-protein complex after treatment with these various agents is largely owing to an induced transition of ColE1 DNA from the supercoiled to the open circular state.

Formation and detection of rna-dna hybrid molecules in cytological preparations

Abstract: A technique is described for forming molecular hybrids between RNA in solution and the DNA of intact cytological preparations. Cells in a conventional tissue squash are immobilized under a thin layer of agar. Next they are treated with alkali to denature the DNA and then incubated with tritium-labeled RNA. The hybrids are detected by autoradiography. The technique is illustrated by the hybridization of ribosomal RNA to the amplified ribosomal genes in oocytes of the toad Xenopus. A low level of gene amplification was also detected in premeiotic nuclei (oogonia).

THE COVALENT STRUCTURE OF AN ENTIRE γG IMMUNOGLOBULIN MOLECULE

Abstract: The complete amino acid sequence of a human gammaG1 immunoglobulin (Eu) has been determined and the arrangement of all of the disulfide bonds has been established. Comparison of the sequence with that of another myeloma protein (He) suggests that the variable regions of heavy and light chains are homologous and similar in length. The constant portion of the heavy chain contains three homology regions each of which is similar in size and homologous to the constant region of the light chain. Each variable region and each constant homology region contains one intrachain disulfide bond. The half-cystines participating in the interchain bonds are all clustered within a stretch of ten residues at the middle of the heavy chains.These data support the hypothesis that immunoglobulins evolved by gene duplication after early divergence of V genes, which specified antigen-binding functions, and C genes, which specified other functions of antibody molecules. Each polypeptide chain may therefore be specified by two genes, V and C, which are fused to form a single gene (translocation hypothesis). The internal homologies and symmetry of the molecule suggest that homology regions may have similar three-dimensional structures each consisting of a compact domain which contributes to at least one active site (domain hypothesis). Both hypotheses are in accord with the linear regional differential of function in antibody molecules.

The nerve growth factor: purification as a 30,000-molecular-weight protein

Abstract: The nerve growth factor protein was purified over 100-fold from adult mouse salivary glands. The first step was a gel filtration on Sephadex G-100 at pH 7.5 of the aqueous gland extract. After gel filtration, most of the NGF activity was eluted in the 80,000-90,000-molecular-weight region (G-100 pool). The G-100 pool was dialyzed at pH 5.0 and fractionated by CM52 cellulose chromatography at pH 5.0. Recovery from CM52 cellulose columns was quantitative for protein and ranged 80-100 per cent for the nerve growth factor activity; the latter was almost completely carried by a protein which did not show any heterogeneity when examined by several analytical tests. The purified nerve growth factor showed an S20,w = 2.43, a D20,w = 7.30 and a 30,000 molecular weight. The over-all recovery was about 45 per cent.

Oncogenes of rna tumor viruses as determinants of cancer

Abstract: Evidence from sero-epidemiological studies and from cell culture studies supports the hypothesis that the cells of many, and perhaps all, vertebrates contain information for producing C-type RNA viruses. It is postulated that the viral information (the virogene), including that portion responsible for transforming a normal cell into a tumor cell (the oncogene), is most commonly transmitted from animal to progeny animal and from cell to progeny cell in a covert form. Carcinogens, irradiation, and the normal aging process all favor the partial or complete activation of these genes. An understanding of how normal cells and normal animals prevent expression of endogenous viral information would appear to offer one of the best hopes for the control of naturally occurring cancers.

Cyclic nucleotide-dependent protein kinases, iv. widespread occurrence of adenosine 3′,5′-monophosphate-dependent protein kinase in various tissues and phyla of the animal kingdom

Abstract: Adenosine 3′,5′-monophosphate-dependent protein kinase activity was found in about thirty sources including many mammalian tissues as well as species representative of eight different invertebrate phyla. The data support a unifying theory for the mechanism of action of adenosine 3′,5′-monophosphate, namely that its many and diverse effects are mediated through activation of tissue-specific protein kinases.

The radioimmunoassay of circulating carcinoembryonic antigen of the human digestive system

Abstract: A radioimmunoassay has been developed for determining the serum levels of carcinoembryonic antigen of the human digestive system in patients with cancer of the colon and rectum The assay is simple to perform and has a high degree of reproducibility and specificity The test detects a concentration of 25 ng of carcinoembryonic antigen per ml of serum and this has provided the first demonstration of a circulating tumor-specific antigen in the sera of cancer patients

On population growth in a randomly varying environment

Abstract: If a population is growing in a randomly varying environment, such that the finite rate of increase per generation is a random variable with no serial autocorrelation, the logarithm of population size at any time t is normally distributed. Even though the expectation of population size may grow infinitely large with time, the probability of extinction may approach unity, owing to the difference between the geometric and arithmetic mean growth rates.

α-KETO ACID DEHYDROGENASE COMPLEXES, X. REGULATION OF THE ACTIVITY OF THE PYRUVATE DEHYDROGENASE COMPLEX FROM BEEF KIDNEY MITOCHONDRIA BY PHOSPHORYLATION AND DEPHOSPHORYLATION

Abstract: This paper reports the discovery that the activity of the multienzyme pyruvate dehydrogenase complex from beef kidney mitochondria is regulated by a phosphorylation-dephosphorylation reaction sequence. The site of this regulation is the pyruvate dehydrogenase component of the complex. Phosphorylation and concomitant inactivation of pyruvate dehydrogenase are catalyzed by an ATP-specific kinase (i.e., a pyruvate dehydrogenase kinase), and dephosphorylation and concomitant reactivation are catalyzed by a phosphatase (i.e., a pyruvate dehydrogenase phosphatase). The kinase and the phosphatase appear to be regulatory subunits of the pyruvate dehydrogenase complex.

Radioimmunoassay for the measurement of adenosine 3′,5′-cyclic phosphate

Abstract: A sensitive and specific radioimmunoassay for adenosine 3′,5′-cyclic phosphate (cyclic AMP) has been developed which allows measurement of the nucleotide in extracts of 5-10 mg of tissue. The radioimmunoassay is sufficiently specific for cyclic AMP to eliminate the need for prior chromatographic separation of the cyclic nucleotide from other tissue nucleotides. The radioimmunoassay system is based upon competition of cyclic AMP with a labeled cyclic AMP derivative of high specific activity for binding sites on an antibody specific for the cyclic nucleotide. Antibody to cyclic AMP was obtained by immunizing rabbits with an antigen prepared by conjugating succinyl cyclic AMP with human serum albumin. A high specific activity derivative of cyclic AMP was prepared by synthesizing succinyl cyclic AMP tyrosine methyl ester (SCAMP-TME) and iodinating the phenolic hydroxyl group of the tyrosine moiety with 125I. Free and antibody-bound 125I-SCAMP-TME were separated by precipitation of the antibody-bound fraction with a second antibody (goat anti-rabbit gamma globulin). Displacement of 125I-SCAMP-TME by unlabeled cyclic AMP when plotted as a semilogarithmic function was linear over a concentration range of 2-100 picomoles. The specificity of the antibody was tested against structurally related nucleotides, nucleosides, and purine bases. All had less than 0.005 per cent of the potency of cyclic AMP in inhibiting 125I-SCAMP-TME binding. The marked differences in affinity of the various cyclic nucleotides to cyclic AMP antibody would suggest that antibodies can be developed for each of the cyclic nucleotides by the principles used in this work.

A difference in the architecture of the surface membrane of normal and virally transformed cells

Abstract: Several tissue culture cell lines that were transformed by a tumor virus have been found to react with an agglutinin, while under identical conditions their untransformed parent cell lines did not agglutinate. Since a short treatment of the parent cell line with low concentrations of proteases exposed the same agglutinin receptor sites in a fashion indistinguishable from the transformed cells, it is proposed that both viral and chemical transformation produce changes in the architecture of the membrane, identical to those of the proteases.

Molecular hybridization of radioactive dna to the dna of cytological preparations

Abstract: A method is presented for detecting the cellular location of specific DNA fractions. The technique involves the hybridization of a radioactive test DNA in solution to the stationary DNA of a cytological preparation. Sites of DNA binding are then detected by autoradiography. Experiments with DNA of the toad Xenopus are described.

Interaction of the carbohydrate-binding protein concanavalin a with normal and transformed cells

Abstract: It has been shown that the carbohydrate-binding protein concanavalin A (ConA) can agglutinate leukemic cells and cells transformed by polyoma virus, simian virus 40, chemical carcinogens, and X-irradiation. This protein did not agglutinate normal cells under the same conditions. The agglutination was reversed by competition with α-methyl-D-glucopyranoside (α-MG), a carbohydrate that strongly binds to ConA, but not by the carbohydrates α-methyl-L-fucopyranoside or N-acetylglucosamine, with no binding or weak binding to ConA. Destruction of the α-MG binding sites of the native protein by removal of bivalent metal ions abolished the agglutination produced by the native protein. The treatment of cells with trypsin resulted in the agglutination of normal cells by ConA and a decrease of agglutinability of transformed cells. When nonagglutinating untransformed 3T3 cells were infected with simian virus 40 and normal rat cells were infected with polyoma virus, the infected cells became agglutinable several days after virus infection. The percentage of cells agglutinated, about 50 per cent, was much higher than the percentage of cells hereditarily transformed. The results indicate that the surface membrane of transformed cells contains sites that interact with the α-MG binding sites of ConA, that such sites can be found on the surface membrane of normal cells after treatment with trypsin, and that the change in the surface structure from normal to transformed occurs in cells that are abortively transformed.

Establishment of functional clonal lines of neurons from mouse neuroblastoma

Abstract: Clonal lines of neurons were obtained in culture from a mouse neuroblastoma. The neuroblastoma cells were adapted to culture growth by the animal-culture alternate passage technique and cloned after single-cell plating. The clonal lines retained the ability to form tumors when injected back into mice. A striking morphological change was observed in the cells adapted to culture growth; they appeared as mature neurons, while the cells of the tumor appeared as immature neuroblasts. Acetylcholinesterase and the enzymes for the synthesis of neurotransmitters, cholineacetylase and tyrosine hydroxylase were assayed in the tumor and compared with brain levels; tyrosine hydroxylase was found to be particularly high, as described previously in human neuroblastomas. The three enzymes were found in the clonal cultures at levels comparable to those found in the tumors. Similarly, there were no remarkable differences between the three clones examined.

On the behavior of a capillary surface in a wedge

Abstract: Estimates above and below are obtained for the height of the equilibrium free surface of a liquid when the liquid partially fills a cylindrical container whose cross section contains a corner with interior angle 2α. The surface is characterized by the condition that its mean curvature be proportional to its height above a reference plane (or, in the case of zero gravity, that the mean curvature be constant), and by the requirement that it meet the container wall with prescribed contact angle γ. It turns out that the qualitative behavior of such a surface near the vertex changes markedly, according as α + γ < ½π, or α + γ ≥ ½π. In the former case, the surface is either unbounded or fails to exist, while in the latter case every such surface is bounded. Some experimental comparisons are indicated, and an application to the problem of describing the mechanism of water rise in trees is discussed. The above results describe a limiting case among corresponding properties that hold for surfaces defined over domains with smooth boundaries. This extension is indicated, as well as a formal extension to n-dimensional surfaces; here the interest centers on the fact that it is the mean curvature of an (n-1)-dimensional boundary element that controls the local behavior of the n-dimensional solution surface.

Evidence that xeroderma pigmentosum cells do not perform the first step in the repair of ultraviolet damage to their dna

Abstract: Xeroderma pigmentosum (XP) is a recessively transmitted disorder of man characterized by increased sensitivity to ultraviolet light. Homozygous, affected individuals, upon exposure to sunlight, sustain severe damage to the skin; this damage is characteristically followed by multiple basal and squamous cell carcinomas and not uncommonly by other malignant neoplasia. A tissue culture cell line was derived from the skin of a man with XP. Our measurements of ultraviolet-induced pyrimidine dimers in cellular DNA show that normal diploid human skin fibroblasts excise up to 70 per cent of the dimers in 24 hours, but that fibroblasts derived from the individual with XP excise less than 20 per cent in 48 hours. Alkaline gradient sedimentation experiments show that during the 24 hours after irradiation of normal cells a large number of single-strand breaks appear and then disappear. Such changes are not observed in XP cells. XP cells apparently fail to start the excision process because they lack the required function of an ultraviolet-specific endonuclease. These findings, plus earlier ones of Cleaver on the lack of repair replication in XP cells, raise the possibility that unexcised pyrimidine dimers can be implicated in the oncogenicity of ultraviolet radiation.

Hydrocarbon distribution of algae and bacteria, and microbiological activity in sediments.

Abstract: The chemical taxonomic relationship of microorganisms has been studied through the hydrocarbon fraction of their chemical constituents. The diagenesis and biological transformations of some hydrocarbons in sediments is suggested, as a result of this information.

Xeroderma pigmentosum: a human disease in which an initial stage of dna repair is defective

Abstract: Homozygous xeroderma pigmentosum fibroblasts cannot repair damage to DNA bases, but can repair damage that involves chain breaks. In xeroderma pigmentosum, therefore, there is a defect in an early step in repair at which base damage is recognized and the polynucleotide chain broken enzymatically (by an endonuclease). Heterozygous fibroblasts repair base damage to normal extents. Carcinogenesis in xeroderma pigmentosum, and perhaps in some normal individuals, may be the result of somatic mutations caused by unrepaired damage.

Solubilization of particulate proteins and nonelectrolytes by chaotropic agents

Abstract: Chaotropic ions (those ions which favor the transfer of apolar groups to water) provide a highly effective means for the resolution of membranes and multicomponent enzymes and for increasing the water solubility of particular proteins and nonelectrolytes. The action of chaotropic agents is related to their effect on the structure and lipophilicity of water.

Neuronal dynamics and axonal flow, iv. blockage of intra-axonal enzyme transport by colchicine

Abstract: The fact that the nucleated center of the nerve cell is the major source of the macromolecular materials required in the maintenance and function of the whole neuron requires the operation of a steady cellulifugal convection of these supplies into and down the nerve fiber. This proximo-distal traffic has been firmly established, but the mechanisms involved in it are still poorly understood. Besides the slow (ca. 1 mm per day) advance of the axonal column as a whole (“axonal flow” in the strict sense), the demonstration of additional, much faster, traffic rates (up to several cm per day) calls for special conduits within the axon (“intra-axonal flow”). To test the possible role of neurotubules (average width:220 A) in this traffic, the drug colchicine, known for its immobilizing effect on microtubules in other types of cells, was locally injected into peripheral nerves. This resulted in a major blockage of the proximo-distal movement of a test enzyme, acetylcholinesterase, into and through the injected zone, the extent of blockage varying with the applied dosage. By analogy, the neurotubules thus seem to be definitely implicated in the motile mechanism of intra-axonal transport. By contrast, the movement of a mitochondrion-associated marker enzyme, diphosphopyridine nucleotide diaphorase, was not perceptibly affected (in the submaximal dosage range), which seems to signify that the proximo-distal shift of mitochondria, for which the slow axonal flow acts as carrier, has gone on uninterruptedly. The experiments thus indicate the possibility of uncoupling the axonal and intra-axonal transport mechanisms.

Negative cooperativity in regulatory enzymes

Abstract: Negative cooperativity has been observed in CTP synthetase, an allosteric enzyme which contains a regulatory site. Thus, the same enzyme exhibits negative cooperativity for GTP (an effector) and glutamine (a substrate) and positive cooperativity for ATP and UTP (both substrates). In the process of the delineation of these phenomena, diagnostic procedures for negative cooperativity were developed. Application of these procedures to other enzymes indicates that negative cooperativity is a characteristic of many of them. These findings add strong support for the sequential model of subunit interactions which postulates that ligand-induced conformational changes are responsible for regulatory and cooperative phenomena in enzymes.

Interaction of insulin with the cell membrane: the primary action of insulin

Abstract: Insulin can be covalently attached to a large polymers of Sepharose through the α-amino group of the N-terminal residue of the B chain, or through the e-amino group of its lysyl residue Such derivatives effectively increase the utilization of glucose, and suppress the hormone-stimulated lipolysis, of isolated fat cells The effects occur with concentrations of insulin-Sepharose that are nearly as low as those of native insulin, and the maximal responses are the same The results indicate that interaction of insulin with superficial membrane structures alone may suffice to initiate transport as well as other metabolic alterations

Calorimetric evidence for the liquid-crystalline state of lipids in a biomembrane

Abstract: Both membranes of Mycoplasma laidlawii and water dispersions of protein-free membrane lipids exhibit thermal phase transitions that can be detected by differential scanning calorimetry. The transition temperatures are lowered by increased unsaturation in the fatty acid residues, but in each case they are the same for membranes and lipids. The transitions resemble those observed for synthetic lipids in the lamellar phase in water, which arise from melting of the hydrocarbon chains within the phospholipid bilayers. Such melts are cooperative phenomena and would be greatly perturbed by apolar binding to protein. Thus the identity of membrane and lipid transition temperatures suggests that in the membranes, as in water, the lipids are in the bilayer conformation in which the hydrocarbon chains associate with each other rather than with proteins. Observations of morphological changes indicate that osmotic imbalance occurs when the membrane transition temperature exceeds the growth temperature, and that for transport processes to function properly the hydrocarbon chains must be in a liquid-like state.

Excited-state two-proton tautomerism in hydrogen-bonded n-heterocyclic base pairs

Abstract: The optical absorption and luminescence spectra of 7-azaindole and its doubly hydrogen-bonded dimer were investigated as a model for the study of electronic interactions in DNA base pairs. It is demonstrated that a biprotonic phototautomerism occurs in the dimer and in suitable ethanol solvates in fluid solvents but that the phenomenon is not observed in a rigid solvent matrix. The normal violet structured fluorescence of 7-azaindole monomer becomes a broad green fluorescence in the tautomer. It is shown that spectral band interchanges, excimer formation, excited-state single-proton transfer, and proton tunneling cannot account for the luminescence change, but that the molecular exciton effect facilitates the cooperative two-proton reversible transfer. It is proposed that biprotonic phototautomerism with molecular environmental sensitivity could provide a mechanism for the initial step in ultraviolet mutagenic effects in DNA.

α-KETO ACID DEHYDROGENASE COMPLEXES, XI. COMPARATIVE STUDIES OF REGULATORY PROPERTIES OF THE PYRUVATE DEHYDROGENASE COMPLEXES FROM KIDNEY, HEART, AND LIVER MITOCHONDRIA

Abstract: The activity of the multienzyme pyruvate dehydrogenase complexes, isolated from mitochondria of beef kidney, beef heart, and pork liver, is regulated by phosphorylation and dephosphorylation. Phosphorylation and concomitant inactivation of each of the three complexes are catalyzed by an ATP-specific kinase, and dephosphorylation and concomitant reactivation are catalyzed by a phosphatase. The phosphatase has been separated from the other component enzymes of each pyruvate dehydrogenase complex, and the three phosphatases are functionally interchangeable. The kinase has been isolated from the beef kidney complex, and it is functional with the beef heart and pork liver complexes. ADP is competitive with ATP, and the ADP effect is more pronounced with the kidney kinase than with the liver and heart kinases. Pyruvate protects strongly the heart and liver pruvate dehydrogenase complexes and, to a lesser extent, the kidney complex against inactivation by ATP. Pyruvate apparently exerts its effect on the pyruvate dehydrogenase component of the complex, rather than on the kinase.

In vitro differentiation of a mouse neuroblastoma.

Abstract: Mouse tumor C1300 has been established in tissue culture. The cells have a round cell morphology in both the subcutaneous tumor and in suspension culture. However, when given a surface on which to attach, they send out processes up to 3 mm in length and assume the morphology of mature neurons. The attached cells are stained by the Bodian silver procedure for neurons, whereas the cells grown in suspension are not. Electron microscopy reveals that the attached cells contain neurofilaments, neurotubules, and densecore vesicles indicative of nerve fibers. Both free-floating and attached cells have tyrosine hydroxylase activity characteristic of sympathetic nervous tissue. Apparently cell attachment can induce morphological differentiation from an anaplastic round cell to a cell which has many properties of a mature neuron.

CHICK CARTILAGE COLLAGEN: A NEW TYPE OF α1 CHAIN NOT PRESENT IN BONE OR SKIN OF THE SPECIES

Abstract: Isolation and chromatography of collagen from chick cartilage revealed an excess of α1 relative to α2-chains. Characterization of the cyanogen bromide peptides from the α1 fraction indicated the presence of two homologous forms of α1. The results suggest that cartilage contains two collagens, one identical to the collagen of skin and bone, the other clearly different requiring the expression of an additional structural gene and possibly containing only α1-chains.

The rate of molecular evolution considered from the standpoint of population genetics

Abstract: The rate of amino acid substitutions in the evolution of homologous proteins is remarkably constant. Furthermore, estimated rates of amino acid substitutions based on comparisons of the alpha hemoglobin chains of various mammals with that of the carp are about the same as those based on comparisons of the carp alpha and mammalian beta or the alpha and beta chains in mammals. These uniformities are regarded as evidence for the hypothesis that a majority of amino acid substitutions that occurred in these proteins are the result of random fixation of selectively neutral or nearly neutral mutations. Two implications of this possibility are discussed: (a) Random gene frequency drift is playing an important role in determining the genetic structure of biological populations and (b) genes in “living fossils” may be expected to have undergone as many DNA base (and therefore amino acid) substitutions as corresponding genes (proteins) in more rapidly evolving species.

Kinetics of memory consolidation: role of amnesic treatment parameters.

Abstract: The consolidation theory states that with the passage of time the engram of a recent learning experience grows increasingly resistant to disruption by amnesic treatment. The time required to reach complete resistance (“consolidation time”) is a controversial issue; current estimates range from 101 to 105 seconds. The present study suggests a parsimonious interpretation of the divergence, namely, that weak amnesic treatments fail to block memory consolidation but do slow its rate, so that post-treatment consolidation inflates the retention scores measured 24 hours later and leads to variably shortened “consolidation times.” This study utilized 2880 neonate chicks trained in a one-trial avoidance paradigm. Retrograde amnesia was induced by treatment with flurothyl (CF3CH2-OCH2CF3) vapor. Apparent “consolidation times” determined by conventional data analysis varied widely as a function of flurothyl concentration and exposure time, ranging from 4 minutes under weak amnesic conditions (0.85% flurothyl; 1-min exposure) to 24 hours under strong conditions (1.7% flurothyl; 8-min exposure). With 1.7% flurothyl, the consolidation half-time was found to be 9.8 hours.

Mixed culture cytopathogenicity: a new test for growth of murine leukemia viruses in tissue culture

Abstract: Tissue culture cells of a Rous virus-induced rat tumor undergo syncytium formation when placed in contact with mouse embryo cell cultures infected with murine leukemia viruses. This phenomenon can be used as a cytopathic end point for isolating and titrating these viruses in tissue culture. The principle should be applicable to detection of leukemia viruses of other species.