bluuebunny/crossref_metadata_2025 huggingface dataset:
Posttranscriptional modification of mRNA conformation: Mechanism that regulates erythromycin-induced resistance
Authors: Horinouchi Sueharu , Weisblum Bernard
Published: May 2006

The nucleotide sequence of a gene in plasmid pE194 responsible for erythromycin-induced resistance, including regulation of the resistance phenotype, is reported. A DNA fragment from plasmid pE194, obtained by digestion with
Taq
I restriction endonuclease, was cloned in
Bacillus subtilis
by using pC194 as the plasmid cloning vector. Erythromycin-resistant, inducible transformant clones containing the
Taq
I fragment A were obtained in which the expression of resistance was similar to that found in the original pE194 background; an interpretative model of the regulation of the erythromycin-resistance determinant is proposed based on the sequence of the
Taq
I A fragment. The cloned
Taq
I A fragment consists of 1442 base pairs and has open reading frames capable of coding for a peptide and a protein containing 19 and 243 amino acids, respectively, referred to as the “leader peptide” and “29,000 protein.” Between the putative transcriptional start site and the ribosome binding site for 29,000-protein synthesis, the promoter region contains four complementary inverted repeat sequences named “1, 2, 3, and 4,” respectively, in which 1 is complementary to 2, 2 is complementary to 3, and 3 is complementary to 4. Sequence 1 encodes the COOH-terminal half of the leader peptide, whereas the ribosome binding site for synthesis of 29,000 protein is sequestered in a loop formed by the association of 3 and 4. The 29,000-protein promoter region does not appear to contain any transcription stop signal. We propose a model for regulation of erythromycin resistance according to which ribosomes engaged in leader peptide synthesis are partially inhibited by optimal inducing (i.e., subinhibitory) concentrations of erythromycin that, in turn, cause an accumulation of these partially inhibited (“stalled”) ribosomes in sequence 1. During induction, the translationally inactive states of association of the inverted repeats, postulated to be 1 plus 2 and 3 plus 4, respectively, are perturbed by a high level of stalled ribosome occupancy in sequence 1, and in the resultant redistribution, 2 associates with 3, freeing 4 and thereby freeing the ribosome binding site sequestered by the association of 3 and 4. Sequence alterations at the 5′ end of the 29,000-protein coding region associated with mutation to constitutive expression have been localized to the inverted complementary repeats, and determination of base changes in eight mutants are all capable of reducing the stability of the postulated stems in a manner consistent with predictions made by the model.
Presence of a flavin semiquinone in methanol oxidase.
Authors: Mincey T , Tayrien G , Mildvan A S , Abeles R H
Published: May 2006

Methanol oxidase from Hansenula polymorpha contains five "red" flavin semiquinones and two oxidized flavins per octamer. Addition of substrate results in the reduction of the two oxidized flavins but does not affect the flavin semiquinones. Enhanced water proton relaxation rates indicate that the unpaired electron of the flavin semiquinones is accessible to the solvent and this accessibility is significantly decreased upon binding of the suicide inhibitor cyclopropanol. In the native enzyme, the semiquinones are not oxidizable by air. All flavins were resolved from the enzyme, and holoenzyme was reconstituted by addition of oxidized flavin. The reconstituted enzyme was catalytically active. The specific activity was 50% that of the original enzyme. It was concluded that the semiquinone is not required for the oxidation of methanol, although it may be present at an otherwise intact site.
Translation and developmental regulation of RNA encoded by the eukaryotic transposable element copia.
Authors: Flavell A J , Ruby S W , Toole J J , Roberts B E , Rubin G M
Published: May 2006

copia-specific RNA was isolated from Drosophila melanogaster tissue culture cells by hybridization of cytoplasmic polyadenylylated RNA to copia DNA immobilized on cellulose. The purified RNA was translated in reticulocyte lysates. One major polypeptide of approximately 51,000 daltons was synthesized in addition to several others between 18,000 and 38,000 daltons. The 51,000-dalton polypeptide and several of the others are encoded by mRNAs of about 2000 nucleotides. The approximate locations on the copia element of the coding sequences for the 51,000-dalton polypeptide and several other proteins were determined by hybrid-arrested translation with copia restriction fragments. The relative abundance of copia-specific RNA was determined at various stages of the Drosophila life cycle. The level of copia-specific RNA is modulated during development of the organism, with the highest level occurring during the larval stages.
Purification of a membrane-associated protein complex required for protein translocation across the endoplasmic reticulum.
Authors: Walter P , Blobel G
Published: May 2006

The capacity of microsomal membranes to translocate nascent presecretory proteins across their lipid bilayer can be largely abolished by extracting them with high ionic strength buffers. It can be reconstituted by adding the salt extract back to the depleted membranes [Warren, G. & Doberstein, B. (1978) Nature (London) 273, 569-571]. Utilizing hydrophobic chromatography, we purified to homogeneity a protein component of the salt extract that reconstitutes the translocation activity of the extracted membranes. This component behaves as a homogeneous species upon gel filtration, ion-exchange chromatography, adsorption chromatography, and sucrose-gradient centrifugation. When examined by polyacrylamide gel electrophoresis in NaDodSO4, six polypeptides with apparent Mr of 72,000, 68,000, 54,000, 19,000, 14,000, and 9000 are observed in about equal and constant stoichiometry, suggesting that they are subunits of a complex. The sedimentation coefficient of 11S is in good agreement with the sum of the Mr of the subunits. The Mr 68,000 and 9000 subunits label intensely with N-[3H]ethylmaleimide. Thus, the reported sulfhydryl group requirement of the translocation activity in the unfractionated extract [Jackson, R. C., Walter, P. & Blobel, G. (1980) Nature (London), 286, 174-176] may be localized to either or both the Mr 68,000 and 9000 subunits of the purified complex.
Receptor structure in the bacterial sensing system.
Authors: Wang E A , Koshland D E
Published: May 2006

The primary receptors for aspartate and serine in bacterial chemotaxis have been shown to be the 60,000-dalton proteins encoded by the tar and tsr genes. The evidence is: (i) overproduction of the tar gene product at various levels by recombinant DNA techniques produces proportionate increases in aspartate binding; (ii) aspartate binding copurifies with [3H]methyl-labeled tar gene product; (iii) antibody to tar and tsr protein fragments precipitates a single species of protein (60,000 daltons) which retains binding capacity and [3H]carboxymethyl label. Partially purified tar gene product can be reconstituted into artificial vesicles and retains aspartate binding and aspartate-sensitive methylation and demethylation. These results show that the aspartate and serine receptors are transmembrane proteins of a single polypeptide chain with the receptor recognition site on the outside of the membrane and the covalent methylation site on the inside.
Inhibition of biological activity of multiplication-stimulating activity by binding to its carrier protein.
Authors: Knauer D J , Smith G L
Published: May 2006

Multiplication-stimulating activity (MSA) produced by Buffalo rat liver cells (BRL-3A) in culture is related to the somatomedin family of growth regulatory polypeptides. MSA will stimulate glucose transport and DNA synthesis in normal chicken embryo fibroblasts (CEF) at concentrations of 10-200 ng/ml. MSA found in BRL-3A-conditioned medium, like the somatomedins in serum, does not exist as the free hormone but is bound to a specific high molecular weight carrier protein. In this report we demonstrate that purified MSA carrier protein (MCP) inhibits the biological activity of MSA on CEF as measured by the stimulation of glucose transport and DNA synthesis. In addition, purified MCP competitively inhibits the binding of 125I-labeled MSA to these cells. In control experiments in which insulin was used as the mitogenic agent, MCP had no effect on these biological responses. These results indicate that the inhibitory effect of MCP is the result of specific interaction with MSA and support the hypothesis that cells may be unresponsive to somatomedins bound to their serum carrier proteins.
Lipid fluidity markedly modulates the binding of serotonin to mouse brain membranes.
Authors: Heron D S , Shinitzky M , Hershkowitz M , Samuel D
Published: May 2006

The binding of [3H]serotonin to mouse brain crude membrane and synaptosomal membrane preparations was investigated as a function of membrane fluidity changes by lipids. The microviscosity (eta) of the synaptic membranes was increased by in vitro incubation with either cholesteryl hemisuccinate or stearic acid, resulting in an up to 5-fold increase in the specific binding of [3H]serotonin. Serotonin binding increased progressively until it reached a maximum at 1.75 relative eta units; then it declined. Fluidization of membrane lipids, by treatment with lecithin or linoleic acid, caused a small but significant decrease in serotonin binding. These observations are compatible with the concept of vertical displacement of membrane proteins, indicating that in the untreated brain tissue the accessibility (Bmax) of serotonin receptor binding sites constitutes only a fraction (about 20%) of the potential binding capacity stored in the membrane. Scatchard plots of [3H]serotonin binding, at different eta values, indicate a continuous change in the binding affinity (Kd) of serotonin to its receptor, concomitant with changes in its accessibility. These results may have important implications for physiological processes in the central nervous system, which are associated with modulation of membrane lipids, such as aging. In addition, the regional heterogeneity and plasticity of receptors may be accounted for by differences in membrane lipid fluidity. It was found here that various brain regions differ markedly in their membrane lipid viscosity.
Synthesis of adult myosin light chains by embryonic muscle cultures.
Authors: Keller L R , Emerson C P
Published: May 2006

Myosin light chain synthesis has been analyzed in cultures of fast and slow muscles from chicken and quail embryos. Synthesis was assayed by [35S]methionine incorporation and two-dimensional electrophoresis of total cell extracts. Our results show that differentiated cultures of embryonic anterior latissimus dorsi and pectoral muscles synthesize proteins that comigrate on two-dimensional gels with the five myosin light chains of adult fast (pectoral) and slow (anterior latissimus dorsi) muscle. Partial proteolytic digestion and peptide analyses further confirm the identity of these proteins as adult light chains. Cultures of dividing myoblasts do not synthesize any of these fiber type isozymes, and synthesis of the isozymes is initiated at myoblast fusion. Also, myogenic clones drived from single myoblasts differentiate to synthesize these five myosin light chains, indicating that individual myoblasts have the potential to express the synthesis of all fiber type light chain isozymes. We conclude that the primary events in muscle differentiation include the initiation of synthesis of the entire set of adult fast and slow myosin light chain isozymes. The developmental and physiological implications of these results for the establishment of fiber type specificity are discussed.
Distribution and activity of endogenous lectin during myogenesis as measured with antilectin antibody.
Authors: Podleski T R , Greenberg I
Published: May 2006

Antibodies to electrolectin, a lectin endogenous to embryonic skeletal muscle, have been used to study the distribution of electrolectin during myogenesis in L6 cells and rat primary muscle cultures. Antibody binding is highest to mononucleated cells and is low to myotubes in both systems. Binding is much lower to fibroblasts in the primary cultures. Binding appears to be on the surface of these cells, although evidence is presented for there being binding on the inside of cells as well. When observed on myotubes, binding is generally associated with highly stained patches and in some instances is near regions where fusion may be occurring, In L6 cells, binding sites can be exposed by treating mononucleated cells with trypsin. These results are discussed in terms of their possible role in myogenesis and synaptogenesis.
Escherichia coli mutator mutants deficient in methylation-instructed DNA mismatch correction.
Authors: Glickman B W , Radman M
Published: May 2006

Our approach to the isolation of DNA mismatch-correction-deficient mutants was based upon the isolation of 2-aminopurine-resistant second-site revertants of Escherichia coli dam- mutants. We isolated such second-site revertants which, when separated from the dam- mutation, have a mutator character of their own. These new mutators all mapped at three known mutator loci, mutH, mutL, and mutS, which exhibit the same mutagenic spectrum as the dam- mutator: increased levels of base substitution and frameshift mutations. The mutator potencies of double and triple mut- mutants suggest that these mutators are involved in the same general mismatch-repair pathway. All these mutations result in a hyper-recombination phenotype, but in four-factor crosses among lambda phages, a specific loss of intragenic recombination (Pam3 X Pam80) was found in mutL and mutS mutants, as would be predicted from the postulated role of mismatch correction in gene conversion and high negative interference phenomena.
Primary structural studies of an H-2L molecule confirm that it is a unique gene product with homology to H-2K and H-2D antigens.
Authors: Coligan J E , Kindt T J , Nairn R , Nathenson S G , Sachs D H , Hansen T H
Published: May 2006

Radiochemical methodology has been used in the isolation and preliminary biochemical characterization of the murine H-2Ld major histocompatibility complex gene product. The radiolabeled molecule was isolated by immunoprecipitation from the glycoprotein fraction of detergent-solubilized H-2d tumor cells. Six major CNBr fragments were isolated from a papain fragment of this molecule; three of the fragments are connected by disulfide bonds. Due to the high degree of homology between major transplantation antigens, it was possible to align the fragments by comparison of their amino acid sequences to that of the H-2Kb gene product. Of the positions available for comparison between H-2Ld and H-2Kb, H-2Dd, and H-2Kd gene products, 61 out of 80 (78%), 45 out of 55 (82%), and 12 out of 15 (80%), respectively, are identical. Differences between the Ld and Kb and Dd molecules are distributed throughout the amino acid sequence. These data indicate that the H-2Ld gene product is a molecular species distinct from, but homologous to, the H-2K and H-2D gene products.
Calcium dependence of phalloidin-induced liver cell death.
Authors: Kane A B , Young E E , Schanne F A , Farber J L
Published: May 2006

The role of Ca2+ in toxic liver cell death was studied with primary cultures of adult rat hepatocytes. Within 1 hr of exposure to phalloidin, a bicyclic heptapeptide isolated from the mushroom Amanita pahlloides, at 50 micrograms/ml, 60--70% of the cells were dead (trypan blue stainable). There was no loss of viability of the same cells exposed to phalloidin in culture medium devoid of Ca2+. A marked structural alteration of the surface of the phalloidin-treated hepatocytes characterized by innumerable evaginations seen by scanning electron microscopy occurred in the presence or absence of Ca2+. Pretreatment of the cells with cytochalasin B at 10 micrograms/ml prevented the surface alteration and the death of the cells in Ca2+ medium. Exposure of the cells to phalloidin in the absence of Ca2+ followed by exposure to cytochalasin B and then to Ca2+ also prevented the cell death. These results suggest a two-step mechanism by which phalloidin causes liver cell death. Initially phalloidin interacts in a Ca2+-independent process with cell membrane-associated actin. The second step is a Ca2+-dependent process that most likely represents an increased influx of Ca2+ across a compromised cell membrane permeability barrier and down the steep concentration gradient that exists between the outside and inside of the cell. These results strengthen the hypothesis that disturbances in Ca2+ homeostasis induced in vivo by a variety of hepatotoxins are causally related to liver cell death.
Development of circadian rhythmicity and light responsiveness in the rat suprachiasmatic nucleus: a study using the 2-deoxy[1-14C]glucose method.
Authors: Fuchs J L , Moore R Y
Published: May 2006

The suprachiasmatic nucleus (SCN) of the hypothalamus is thought to play a critical role in circadian rhythm generation and entrainment to the light/dark cycle. In adult rats, the SCN shows a circadian rhythm in metabolic activity level as indicated by 2-deoxy[1-14C]glucose uptake. In the present study, the development of this rhythm was investigated. No diurnal difference in uptake was evident in fetal rats 1--2 days before birth. A significant diurnal difference in SCN 2-deoxyglucose uptake was preseno light at night increased the SCN metabolic levels. According to previous studies, on day 1 the SCN is poorly developed and contains few synapses. At this time the retinohypothalamic tract has not yet developed. We found progressive functional maturation of the SCN through day 21, when the rhythm and light responsiveness resembled those of adult rats.
Plasmids containing insertion elements are potential transposons.
Authors: Ohtsubo E , Zenilman M , Ohtsubo H
Published: May 2006

We studied in vivo recombination between the plasmid pHS1, a temperature-sensitive replication mutant carrying tetracycline resistance, and pSM1, a small plasmid carrying one copy of the insertion element IS1. Recombinant plasmids were found by selection for tetracycline resistance at 42 degrees C. Their formation was independent of recA function. Analysis of the physical structures of various recombinant DNA molecules with electron microscopy and restriction endonucleases revealed that pSMI was integrated at its IS1 into numerous sites on pHS1, giving rise to a duplication of IS1 in the same orientation at both junctions. Nucleotide sequence analysis of recombinant plasmids and their parental plasmid DNA revealed that nine nucleotides at a target site were duplicated at the junction of each IS1. This phenomenon implies that plasmids containing a translocatable DNA element can be potential transposons.
Evidence for involvement of proline cis-trans isomerization in the slow unfolding reaction of RNase A.
Authors: Garel J R
Published: May 2006

The three exposed tyrosines of RNase A have been converted to nitrotyrosines by reaction with tetranitromethane, and the changes in the ionization properties of these nitrotyrosines have been used to follow the kinetics of unfolding of the nitrated protein. It is found that the nitrotyrosines not only are sensitive to the overall disruption of the protein structure, which occurs in a faster reaction, but also serve as reporter groups for the slower reaction which takes place in the unfolded state. This slower reaction corresponds to the formation of the slow-refolding species of the unfolded protein. The kinetic properties of the slower reaction--guanidine-dependence of the rate and activation ethalpy--are similar to those of the proline cis-trans isomerization in a model peptide determined in the same conditions. It is concluded that proline cis-trans isomerization is indeed the rate-limiting factor for the formation of the slow-refolding species. Because the influence of proline cis-trans isomerization on the properties of the nitrotyrosines in the unfolded protein is probably due to a local effect, it is suggested that most of the optical changes observed during this slow unfolding reaction arise from the effect of the cis-trans isomerization of the Asn113-Pro114 bond on the properties of nitrotyrosine 115.
Fluorescence staining of the actin cytoskeleton in living cells with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin.
Authors: Barak L S , Yocum R R , Nothnagel E A , Webb W W
Published: May 2006

An active fluorescent derivative of the actin-binding mushroom toxin phallacidin has been synthesized. Convenient methods were developed to stain actin cytoskeletal structures in living and fixed cultured animal cells and actively streaming algal cells. Actin binding specificity was demonstrated by competitive binding experiments and comparative staining of well-known structures. Large populations of living animal cells in culture were readily stained by using a relatively mild lysolecithin permeabilization procedure facilitated by the small molecular size of the label. Actin in animal cells was stained stress fibers, ruffles, the cellular geodome, and in diffuse appearing distributions apparently associated with the plasma membrane. Staining of actin cables in algae with nitrobenzoxadiazole (NBD)-phallacidin did not inhibit cytoplasmic streaming. NBD-phallacidin provides a convenient actin-specific fluorescent label for cellular cytoskeletal structures with promise for use in studies of actin dynamics in living systems.
Mechanisms of triboluminescence
Authors: Lin S. H. , Wutz D. , Ho Z. Z. , Eyring H.
Published: May 2006

The purpose of this paper has been to develop the theoretical treatment of the triboexcitation mechanisms due to the electric field effect and the pressure effect and to show how to analyze the triboluminescent spectra to determine the external factors that affect or induce the triboluminescence.
Purification from rat sarcolemma of the saxitoxin-binding component of the excitable membrane sodium channel.
Authors: Barchi R L , Cohen S A , Murphy L E
Published: May 2006

The saxitoxin-binding component (SBC) of the excitable membrane sodium channel has been solubilized and purified from rat skeletal muscle sarcolemma. Phospholipid was required in mixed micelles with detergent for stability of the mammalian SBC. Even at optimal detergent-to-phospholipid ratio, the solubilized SBC showed significant temperature-dependent loss of specific toxin binding with time, necessitating maintenance of low temperatures during purification. Characteristics of saxitoxin binding to the solubilized material closely resembled those seen in intact membranes. A weak anion-exchange column was synthesized; it provided rapid 10- to 20-fold purification of the solubilized SBC. Additional necessary purification was obtained by chromatography on immobilized wheat germ agglutinin. Specific saxitoxin-binding activity of the purified material averaged approximately 1500 pmol of saxitoxin bound per mg of protein. Three bands were present in this material on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The purified material sedimented on a sucrose gradient with an apparent s20,w of 9.9 S.
Primary structure of elongation factor Tu from Escherichia coli.
Authors: Arai K , Clark B F , Duffy L , Jones M D , Kaziro Y , Laursen R A , L'Italien J , Miller D L , Nagarkatti S , Nakamura S , Nielsen K M , Petersen T E , Takahashi K , Wade M
Published: May 2006

The amino acid sequence of elongation factor Tu (EF-Tu) from Escherichia coli has been determined. EF-Tu is a single-chain polypeptide composed of 393 amino acids (Mr 43,225 for the species bearing COOH-terminal serine). The NH2-terminal serine is acetylated, and lysine-56 is partially methylated. The sites of facile tryptic cleavage are at arginines 44 and 58 and at lysine-263. The cysteinyl residues associated with aminoacyl-tRNA and guanosine nucleotide binding activities are residues 81 and 137, respectively. The COOH-terminal amino acid is heterogenous in that analyses of the COOH-terminal peptides isolated from different EF-Tu preparations gave position 393 as glycine and serine in ratios (Gly/Ser) ranging from about 0.7 to 3.
Structurally unique basic protein coextracted with histones from calf thymus chromatin.
Authors: Jenson J C , Chin-Lin P , Gerber-Jenson B , Litman G W
Published: May 2006

A histone-like protein that is rich in alanine and lysine (protein AK) has been obtained in homogeneous form by high-resolution gel filtration of H2SO4 extracts of calf thymus chromatin. Protein AK: (i) migrates as a single band in polyacrylamide gel electrophoresis in both acetic acid/urea and sodium dodecyl sulfate; (ii) is a basic protein; (iii) lacks tryptophan; (iv) is not extracted from chromatin with 0.35 M NaCl; and (v) is not soluble in 0.75 M HClO4. Protein AK is distinguished from the high-mobility group (HMG) proteins on the basis of these latter solubility characteristics and from the histones and protein A24 on the basis of amino acid composition and distribution of tryptic peptides in two-dimensional chromato-electrophoresis. In addition, protein AK is distinguished from the HMG proteins, the histones, and protein A24 on the basis of its mobility in two polyacrylamide gel electrophoresis systems. The amino acid composition of protein AK resembles that of HU, a basic DNA-binding protein found in Escherichia coli.
Identification of regulatory sequences in the prelude sequences of an H2A histone gene by the study of specific deletion mutants in vivo.
Authors: Grosschedl R , Birnstiel M L
Published: May 2006

Conserved DNA sequence elements of putative regulatory functions were deleted from the prelude region of a sea urchin H2A histone gene. For this, the wild-type H2A gene of the 6-kilobase histone DNA repeat unit was replaced by various mutant H2A genes by cloning. The effects of the manipulation on H2A mRNA synthesis were studied by injection of the mutant DNAs into centrifuged Xenopus oocytes. The unmanipulated H2B gene residing within the same repeat unit provided a suitable internal control for these studies. Deletion of the T-A-T-A-A-A-T-A motif, once thought to be the functional equivalent of the bacterial Pribnow box, did not abolish transcription of the gene; instead, a number of novel mRNA 5' termini were generated. We argue that the T-A-T-A-A-A-T-A motif is a specificity element, a selector of eukaryotic gene transcription. Deletion of the "cap-sequence," 5' pyrimidine-C-A-T-T-C-purine 3' and most of the mRNA leader sequence did not abolish transcription but created yet another mRNA 5' terminus. In contrast to these deletions, which are both down-mutations, deletion of H2A gene-specific conserved DNA sequences upstream from the T-A-T-A-A-A-T-A motif enhanced mRNA synthesis. A hypothesis for the function of these DNA sequences as eukaryotic promoter elements is discussed.
Relationships among chromatid interchanges, sister chromatid exchanges, and meiotic recombination in
Drosophila melanogaster
Authors: Gatti Maurizio , Pimpinelli Sergio , Baker Bruce S.
Published: May 2006

Repair- and recombination-defective mutations at two loci (
mei-9
and
mei-41
) of
Drosophila melanogaster
have been examined for their effects on the induction of chromosome aberrations by x-rays and the formation of sister chromatid exchanges (SCEs). Irradiation of larval neuroblast cells during the S phase with x-rays showed that mutants at both of these loci are about 10 times more sensitive than wild type to the induction of chromosome aberrations. The pattern of induced aberrations was characteristic for each mutant locus: in cells bearing
mei-9
mutations most breaks were chromatid deletions, whereas in the presence of
mei-41
mutations similar frequencies of chromatid and isochromatid deletions were observed. Furthermore, chromatid interchanges could not be induced in cells carrying
mei-9
alleles; therefore these mutations define a step necessary for chromatid rejoining.
mei-41
alleles also define a function involved in the formation of chromatid interchanges; total exchanges were less frequent than expected from nonmutant controls; and the proportion of exchanges arising by symmetrical rejoining was markedly reduced. These data indicate that chromatid and isochromatid deletions have different molecular steps in their formation, and that different molecular mechanisms are also involved in the symmetrical and unsymmetrical rejoining in chromatid interchanges. Neuroblast cells of larvae bearing
mei-9
and
mei-41
alleles were also treated for 13 hr with 5-bromodeoxyuridine at 9 μg/ml in order to differentiate sister chromatids for the scoring of SCEs. Whereas
mei-41
had a normal level of SCEs,
mei-9
exhibited a frequency of SCEs that was about 70% that of the control. Because both
mei-9
and
mei-41
mutations result in defective meiotic recombination, these data suggest that they define steps shared by symmetrical interchange formation and meiotic recombination that do not participate in the formation of most SCEs.
Nucleic acid-specific suppressor T cells.
Authors: Borel Y , Young M C
Published: May 2006

The concept of using cell-bound antigens as tolerogen was applied to nucleic acid. Nucleoside was linked directly to spleen cell suspensions. Intravenous administration of nucleoside coupled to isogeneic spleen cells into mice generated suppressor cells that diminished the formation of antibody-forming cells either to a T-dependent antigen in vivo or to a T-independent antigen in vitro. Suppressor cells were nucleoside specific, but the specificity of immune suppression seems to be somewhat broader than that of tolerance to a single nucleoside. The ability to raise nucleic acid-specific suppressor T cells may have implications for both the pathogenesis and treatment of systemic lupus erythematosus.
Specificity of human antibodies to oncovirus glycoproteins: recognition of antigen by natural antibodies directed against carbohydrate structures.
Authors: Snyder H W , Fleissner E
Published: May 2006

Antibodies in human sera from healthy individuals were shown to be reactive with highly purified 70,000-dalton envelope glycoprotein (gp70) of the simian sarcoma virus-simian sarcoma-associated virus (SSV-SSAV) complex in radioimmunoprecipitation assays under certain conditions. The specificity of the reaction was analyzed in absorption tests with normal human serum proteins, assays of viral gp70 antigenicity after exposure to exo- and endoglycosidases or trypsin, and carbohydrate hapten inhibition studies. On the basis of the results obtained in these experiments we have concluded that immune recognition of SSV-SSAV gp70 can be mediated by naturally occurring heterophil antibodies in human sera that are reactive by virtue of binding to the carbohydrate moiety of the viral gp70 molecules. The results are consistent with the idea that the antibodies in question are elicited as a result of exposure to many natural substances possessing widely crossreacting antigens and are not a result of widespread infection of man with replication-competent oncoviruses.
Choline accumulation by photoreceptor cells of the rabbit retina.
Authors: Masland R H , Mills J W
Published: May 2006

Photoreceptor cells of the rabbit retina accumulate choline from the extracellular environment by an overall process that has a high affinity for choline. These cells do not synthesize acetylcholine; instead, the choline taken up is incorporated into phosphorylcholine and eventually phospholipid. A mechanism for efficient choline accumulation is presumably concomitant to the photoreceptor cell's synthesis of large amounts of membrane for outer segment membrane renewal. Its existence in the photoreceptor cell supports previous evidence that high-affinity choline uptake is not confined to neurons that release acetylcholine, but may be present wherever large amounts of choline are required.
Probable precursors of [Leu]enkephalin and [Met]enkephalin in adrenal medulla: peptides of 3-5 kilodaltons.
Authors: Kimura S , Lewis R V , Stern A S , Rossier J , Stein S , Udenfriend S
Published: May 2006

Adrenal chromaffin granules contain at least 10 peptides, ranging in size from 3 to 5 kilodaltons, that yield, upon digestion with trypsin, peptides that show specific binding to opiate receptors. All are distinctly different from beta-endorphin. Two of these peptides have been purified to homogeneity and subjected to chemical analysis. One is apparently a [Met]enkephalin precursor containing two copies of the [Met]enkephalin sequence. The other peptide contains both [Leu]enkephalin and [Met]enkephalin sequences and is presumably a common precursor of the two forms of enkephalin.
Geometrical model for the energy of semicoherent interphase interfaces
Authors: Ecob Roger C. , Ralph Brian
Published: May 2006

The basis for the considerations given in this paper is the O-lattice description of crystalline interfaces of Bollmann. In the development of his approach presented here, all possible interfacial planes between two crystal phases having a defined orientation relationship are considered. The energies of these interfaces are then computed in terms of the energies of the primary intrinsic dislocations. A number of modeling interactions are incorporated into this approach, and a better agreement with experimental data is thus obtained.
Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis.
Authors: Hershko A , Ciechanover A , Heller H , Haas A L , Rose I A
Published: May 2006

The heat-stable polypeptide ATP-dependent proteolysis factor 1 (APF-1) of the reticulocyte proteolytic system forms covalent compounds with proteins in an ATP-requiring reaction. APF-1 and lysozyme, a good substrate for ATP-dependent proteolysis, form multiple conjugates, as was shown by comigration of label from each upon gel electrophoresis. Multiple bands were also seen with other substrates of the ATP-dependent proteolytic system, such as globin or alpha-lactalbumin. Analysis of the ratio of APF-1 to lysozyme radioactivities and of the molecular weights of the bands indicated that they consist of increasing numbers of the APF-1 polypeptide bound to one molecule of lysozyme. The covalent linkage is probably of an isopeptide nature, because it is stable to hydroxylamine and alkali, and polylysine is able to give conjugates of APF-1. Removal of ATP after formation of the 125I-labeled APF-1 conjugates with endogenous proteins caused the regeneration of APF-1, indicating presence of an amidase. This reaction is thought to compete with proteases that may act on APF-1-protein conjugates, especially those containing several APF-1 ligands. A sequence of reactions in which the linkage of APF-1 to the substrate is followed by the proteolytic breakdown of the substrate is proposed to explain the role of ATP.
Reconstitution of functional membrane-bound acetylcholine receptor from isolated Torpedo californica receptor protein and electroplax lipids.
Authors: Gonzalez-Ros J M , Paraschos A , Martinez-Carrion M
Published: May 2006

Purified acetylcholine receptor and total lipids, both extracted from Torpedo californica electroplax, were utilized to reconstitute chemically excitable membrane vesicles. Reconstitution was achieved by dialysis of the extraction detergent, octyl beta-D-glucoside from protein/lipid incubation mixtures. The reconstituted preparations could be fractionated by sucrose density gradient centrifugation and consisted of vesicular structures visible in electron micrographs. In addition, the reconstituted vesicles exhibited;the following properties characteristic of native receptor-enriched membranes: (i) an external distribution of alpha-bungarotoxin-binding sites, (ii) a time-dependent binding of alpha-bungarotoxin that is depressed by preincubation with the cholinergic agonist carbamoylcholine ("desensitization"), (iii) an ability to retain 22Na+ that is lost in the presence of detergents or gramicidin A, and (iv) a carbamoylcholine-induced acceleration of 22Na+ efflux that can be blocked by alpha-bungarotoxin. The purified acetylcholine receptor that was utilized in the reconstitution experiments apparently does not require other protein components for ligand recognition or ion translocation.
Mode of inhibition of electron transport by orthophenanthroline in chromatophores and reaction centers of
Rhodopseudomonas sphaeroides
Authors: Vermeglio André , Martinet Teresina , Clayton Roderick K.
Published: May 2006

In
Rhodopseudomonas sphaeroides
, light causes the transfer of electrons from bacteriochlorophyll to ubiquinone in the photochemical reaction centers. Electrons from this “primary” ubiquinone move on to a secondary ubiquinone. We have studied the manner in which
o
-phenanthroline inhibits the transfer of electrons from primary to secondary ubiquinone in chromatophores (intracytoplasmic membrane fragments) and isolated reaction centers of
Rp. sphaeroides
. The formation of anionic semiquinones, both primary and secondary, is signaled by an absorption band at 450 nm and by band shifts of bacteriopheophytin and bacteriochlorophyll in the near infrared. The pattern of band shifts is different for primary and secondary semiquinone, allowing us to distinguish which quinone has become semireduced. This point was established for isolated reaction centers [Vermeglio, A. & Clayton, R. K. (1977)
Biochim. Biophys. Acta
461, 159-165] and is confirmed here for chromatophores. By adding
o
-phenanthroline at various times during a sequence of actinic light flashes and monitoring the resulting optical absorbance changes, we have found that
o
-phenanthroline inhibits electron transfer from primary semiquinone to secondary quinone if the latter is in its oxidized form, but not if the latter is semireduced. Our findings can explain the decay kinetics of delayed fluorescence from bacteriochlorophyll in
Rp. sphaeroides
as measured by R. P. Carithers and W. W. Parson [(1976)
Biochim. Biophys. Acta
440, 215-232].
Mapping of the two overlapping genes for polypeptides NS1 and NS2 on RNA segment 8 of influenza virus genome.
Authors: Lamb R A , Choppin P W , Chanock R M , Lai C J
Published: May 2006

RNA segment 8 of the influenza virus genome is unique in coding for two polypeptides, NS1 (Mr, approximately 25,000) and NS2 (Mr, approximately 11,000). These polypeptides are synthesized from separate mRNA species. By using cloned DNA derived from RNA segment 8 (NS DNA) the two mRNAs have been mapped on segment 8 by hybridization of mRNAs with restriction endonuclease fragments of the DNA and nuclease S1 digestion methods. These data indicate that the body of the NS1 mRNA (approximately 850 nucleotides) maps at 0.05-0.95 units of the cloned NS DNA and the body of the NS2 mRNA (approximately 340 nucleotides) maps at 0.59-0.95 unitssuggesting that the two mRNAs are 3' coterminal and share the same poly(A) addition site. These positions of the mRNAs on the viral genome segment were confirmed in hybrid-arrested translation experiments using fragments of the cloned NS DNA to inhibit the synthesis in vitro of NS1 or NS2 polypeptides. In addition, in these translation experiments the use of certain DNA fragments resulted in premature termination of the NS1 polypeptide. From these data, it could be estimated that the termination of translation of NS1 is at approximately 0.76 map unit. Thus, the coding regions of the two mRNAs overlap by approximately 144-159 nucleotides, the equivalent of approximately 48-53 amino acids. Peptide mapping experiments indicated that polypeptides NS1 and NS2 do not share methionine- or leucine-containing tryptic peptides. The results obtained indicate the translation of the NS2 mRNA occurs in a reading frame different from that used for NS1.
Photodestruction of acetylcholinesterase.
Authors: Bishop W H , Henke L , Christopher J P , Millar D B
Published: May 2006

Ultraviolet irradiation of 11S acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) produces a loss of tryptophan fluorescence which is best described as the sum of two separable first-order processes, one much more rapid than the other. In addition, the enzyme undergoes an all-or-none inactivation that is monotonically first order. Simultaneous with activity loss, photoscission takes place and results in a molecular weight drop of 1 x 10(5); this decrease is first order with a rate constant identical to that for enzymatic inactivation. These processes are accompanied by apparent conformational changes, as shown by circular dichroic and difference absorption spectra. The relative photochemical inactivation efficiency of incident light is unity when corrected for the wavelength dependence of fluorescence excitation, which is consistent with an efficient Förster resonance transfer of energy among the aromatic chromophores. The extreme sensitivity of acetylcholinesterase to photodestruction upon photon absorption and the several events that follow it not only suggest that these findings might be a basis for a useful molecular probe of the structure of this enzyme, but also indicate that additional care should be taken when conducting spectroscopic studies in the UV region.
L factor that is required for beta-galactosidase synthesis is the nusA gene product involved in transcription termination.
Authors: Greenblatt J , Li J , Adhya S , Friedman D I , Baron L S , Redfield B , Kung H F , Weissbach H
Published: May 2006

The DNA-dependent in vitro synthesis of Escherichia coli beta-galactosidase requires the presence of a soluble protein referred to as L factor [Kung, H., Spears, C. & Weissbach, H. (1975) J. Biol. Chem. 250, 1556-1562]. In the present study, comparison of physical, immunological, and biological properties shows that L factor is the product of the E. coli nusA gene. The nusA gene product is known to interact with bacteriophage lambda N gene protein and to prevent premature termination of transcription from the early lambda promoters. Our results suggest that premature transcription termination in the lac operon of E. coli may also be overcome by the nusA protein.
Identification of a region susceptible to proteolysis in myosin subfragment-2.
Authors: Lu R C
Published: May 2006

Comparison of the NH2-terminal sequence of myosin short subfragment-2 (Mr of subunit = 37,000) and long subfragment-2 (Mr of subunit = 59,000) demonstrates that the former represents the NH2-terminal portion of the latter and suggests that the hinge region in myosin rod is in the COOH-terminal portion of the long subfragment-2.
Path of the polypeptide in bacteriorhodopsin.
Authors: Engelman D M , Henderson R , McLachlan A D , Wallace B A
Published: May 2006

An attempt has been made to fit the amino acid sequence of bacteriorhodopsin to the three-dimensional density map of the molecule. First, seven segments of the sequence were selected as being probable transmembrane alpha helices. Then each of the 5040 possible ways of fitting these seven segments into the seven regions of helical density in the map were evaluated based on the criteria of connectivity of the nonhelical link regions, charge neutralization, and total scattering density per helix. A single model that may be experimentally tested emerged as the most probable.
Direct transfer of cloned genes from bacteria to mammalian cells.
Authors: Schaffner W
Published: May 2006

Induction of a virus infection by cloned simian virus 40 DNA was chosen as a test system to detect transfer of genes from bacteria to cultured mammalian cells. Escherichia coli cells containing a recombinant plasmid with three tandem inserts of simian virus 40 DNA were able to infect CV-1 monkey cells under various conditions. The gene transfer was resistant to DNase I and therefore seems not to occur via free DNA but most likely via uptake of whole bacteria, followed by release of plasmid DNA and generation of infectious circular simian virus 40 DNA in a recombination-excision process. Spontaneous transfer was found to be infrequent, 4 x 10(9) bacteria yielding one infection per 10(7) monkey cells. The frequency was greatly increased by adding bacteria as a calcium phosphate coprecipitate or by fusion of lysozyme-treated bacteria (protoplasts) with monkey cells in the presence of polyethylene glycol. With the latter technique, 10(4) protoplasts gave rise to one infection per 15 monkey cells. Experiments with other cell lines of human, monkey, and mouse origin, and also with bacteria harboring another recombinant plasmid, indicate that DNA transfer from bacteria to mammalian cells is a general phenomenon.
In vivo or in vitro treatments with anti-I-J alloantisera abolish immunity to AKR leukemia.
Authors: Meruelo D , Flieger N , Smith D , McDevitt H O
Published: May 2006

This paper provides evidence for the involvement of immune mechanisms in conferring resistance to a spontaneous AKR leukemia. It is shown that genes in the B, J, or E subregions of the H-2 complex confer resistance to a spontaneously arisen, tissue culture-adapted AKR thymoma, BW5147. A direct correlation is demonstrated between survival to injected BW5147 cells and humoral responsiveness in various hybrids obtained from crosses of AKR mice and C57BL/10 or C3H/DiSn derived congeneic strains differing at H-2. Cellular immunity appears to play no role in resistance to the proliferation of tumor cells. It is further established that development of effective humoral immunity depends on B cells and Ly-1+, 2-, 3- helper T-cells bearing the I-Jk phenotype. These findings seem directly applicable to the spontaneous disease, and results of studies using transformed cells from an overtly leukemic AKR mouse parallel those obtained using BW1547 cells.
Plasma membrane polypeptides of resident and activated mouse peritoneal macrophages.
Authors: Yin H L , Aley S , Bianco C , Cohn Z A
Published: May 2006

With the lactoperoxidase/glucose oxidase-catalyzed iodination method, we have identified at least 19 exteriorly disposed plasma membrane polypeptides on mouse peritoneal macrophages, with molecular weights ranging from 12,000 to 290,000. Resident and inflammatory macrophages could be distinguished by qualitative and quantitative differences in the display of selected polypeptides, although the overall banding patterns were similar. Some of the labeled polypeptides were identified by immunoprecipitation.
Crystalline ribosomes are present in brains from senile humans.
Authors: O'Brien L , Shelley K , Towfighi J , McPherson A
Published: May 2006

Paracrystalline inclusions known as Hirano bodies characteristically appear in the hippocampal region of the brains of humans exhibiting senile and presenile dementias as well as several other neurodegenerative diseases. We present evidence that the currently accepted model for those structures based on alternating filament sheets is not correct, but that Hirano bodies are stacked sheets of membrane-bound ribosomal particles derived from partially degraded rough endoplasmic reticulum or Nissl substance. Using fluorescence staining with acridine orange and ethidium bromide, were have shown that the bodies contain RNA. Spatial filtering of electron micrographs by Fourier techniques shows that the individual particles that make up the arrays have a characteristic shape previously reported for the large subunit of eukaryotic ribosomes. The storage of these ribosomal particles in inclusion bodies may indicate a quiescent state of protein synthesis in the cells. This withdrawal of synthetic mechanisms in the hippocampus may have significant consequences in the loss of ability to consolidate short-term to long-term memory.