bluuebunny/crossref_metadata_2025 huggingface dataset:
A measure of the similarity of sets of sequences not requiring sequence alignment.
Authors: Blaisdell B E
Published: May 2006

Determination of first- and second-order Markov chain homogeneity of sets of nuclear eukaryotic DNA sequences, both coding and noncoding, finds similarities imperceptible to the standard Needleman-Wunsch base matching or dot-matrix algorithms. These measures of the similarities of the distributions of adjacent pairs or triplets are in agreement with accepted evolutionary-tree topologies. Hierarchical clustering of the distributions of doublets of 30 miscellaneous coding sequences gives clusters in reasonable agreement with accepted biological classifications. In addition to similarity by homology, there is also observed similarity of disparate genes in the same organism--for example, all three disparate yeast genes (two enzymes and actin) form a well-distinguished cluster.
In vitro splicing of influenza viral NS1 mRNA and NS1-beta-globin chimeras: possible mechanisms for the control of viral mRNA splicing.
Authors: Plotch S J , Krug R M
Published: May 2006

In influenza virus-infected cells, the splicing of the viral NS1 mRNA catalyzed by host nuclear enzymes is controlled so that the steady-state amount of the spliced NS2 mRNA is only 5-10% of that of the unspliced NS1 mRNA. Here we examine the splicing of NS1 mRNA in vitro, using nuclear extracts from HeLa cells. We show that in addition to its consensus 5' and 3' splice sites, NS1 mRNA has an intron branch-point adenosine residue that was functional in lariat formation. Nonetheless, this RNA was not detectably spliced in vitro under conditions in which a human beta-globin precursor was efficiently spliced. Using chimeric RNA precursors containing both NS1 and beta-globin sequences, we show that the NS1 5' splice site was effectively utilized by the beta-globin branch-point sequence and 3' splice site to form a spliced RNA, whereas the NS1 3' splice site did not function in detectable splicing in vitro, even in the presence of the beta-globin branch-point sequence or in the presence of both the branch-point sequence and 5' exon and splice site from beta-globin. With the chimeric precursors that were not detectably spliced, as with NS1 mRNA itself, a low level of a lariat structure containing only intron and not 3' exon sequences was formed. The inability of the consensus 3' splice site of NS1 mRNA to function effectively in in vitro splicing suggests that this site is structurally inaccessible to components of the splicing machinery. Based on these results, we propose two mechanisms whereby NS1 mRNA splicing in infected cells is controlled via the accessibility of its 3' splice site.
Erythrocyte homeostasis: antibody-mediated recognition of the senescent state by macrophages.
Authors: Singer J A , Jennings L K , Jackson C W , Dockter M E , Morrison M , Walker W S
Published: May 2006

We tested the hypothesis that the accumulation of bound autologous antibody on a "senescent epitope" identifies aged erythrocytes for phagocytic removal by macrophages. Erythrocytes were collected from mice maintained on a hypertransfusion protocol designed to yield cells of defined age. The mouse erythrocytes were assayed for the presence of bound antibody by measuring their susceptibility to ingestion by macrophages from mouse peritoneal exudates and by flow cytofluorometry. Both assays disclosed that only the oldest mouse erythrocytes bore detectable levels of antibody. Flow cytofluorometric analysis revealed that the frequency distribution of IgG isotypes bound to the cells reflected their levels in normal serum. Finally, treatment with trypsin abolished the ability of the macrophages to ingest erythrocytes aged in vivo. These findings support the hypothesis that antibody mediates the clearance of senescent mouse erythrocytes from the circulation and demonstrate that the presence of a trypsin-sensitive recognition structure on macrophages is an essential requirement in this homeostatic process.
Efficient
in vitro
import of a cytosolic heat shock protein into pea chloroplasts
Authors: Lubben Thomas H. , Keegstra Kenneth
Published: May 2006

In order to further our understanding of the targeting of nuclear-encoded proteins into intracellular organelles, we have investigated the import of chimeric precursor proteins into pea chloroplasts. Two different chimeric precursor proteins were produced by
in vitro
expression of chimeric genes. One chimeric precursor contained the transit peptide of the small subunit of soybean ribulose 1,5-bisphosphate carboxylase and the mature peptide of the same protein from pea. The second contained the same transit peptide plus 13 amino acids of the pea mature peptide fused to a cytosolic heat shock protein. The extent of import and binding of the two chimeric proteins was examined by using quantitative assays and was compared to the import of pea small subunit precursor. Both precursor proteins imported well into pea chloroplasts, although the extent of import observed with the chimeric small-subunit-heat shock precursor was less than that observed with the soybean-pea small subunit precursor. The heat shock protein alone did not import into nor bind to chloroplasts. The binding of both the chimeric small-subunit-heat shock protein and the soybean-pea small subunit precursor to chloroplasts was physiologically significant, as shown by the fact that when chloroplasts with bound precursors were isolated, these bound precursors could subsequently be imported.
Substitution of a serine residue for proline-87 reduces catalytic activity and increases susceptibility to proteolysis of Escherichia coli adenylate kinase.
Authors: Gilles A M , Saint-Girons I , Monnot M , Fermandjian S , Michelson S , Bârzu O
Published: May 2006

Amino acid analysis, HPLC separation of trypsin digests, and sequence analysis showed that the thermosensitivity of the adenylate kinase (EC 2.7.4.3) from Escherichia coli K-12 strain CR341 T28 results from substitution of a serine residue for proline-87 in the wild-type enzyme. This mutation is accompanied by decreased affinity for nucleotide substrates and decreased catalysis. Circular dichroism spectroscopy showed a significant change of the secondary structure. This mainly corresponds to a reduction in alpha-helix content (39%) of mutant protein as compared to wild-type adenylate kinase (50%). Altered conformation of thermosensitive adenylate kinase was also manifested by an increase in susceptibility to proteolysis by trypsin. Ap5A and ATP, known to induce important conformational changes in eukaryotic adenylate kinase(s), protected the mutant enzyme against inactivation by trypsin. This seems to indicate that the "loosening" of the three-dimensional structure of E. coli adenylate kinase by proline----serine substitution is largely compensated for when an enzyme X ATP or enzyme X Ap5A complex is formed.
Autogenous regulation of splicing of the transcript of a yeast ribosomal protein gene.
Authors: Dabeva M D , Post-Beittenmiller M A , Warner J R
Published: May 2006

The gene for a yeast ribosomal protein, RPL32, contains a single intron. The product of this gene appears to participate in feedback control of the splicing of the intron from the transcript. This autogenous regulation of splicing provides a striking analogy to the autogenous regulation of translation of ribosomal proteins in Escherichia coli.
Affinity purification of sequence-specific DNA binding proteins.
Authors: Kadonaga J T , Tjian R
Published: May 2006

We describe a method for affinity purification of sequence-specific DNA binding proteins that is fast and effective. Complementary chemically synthesized oligodeoxynucleotides that contain a recognition site for a sequence-specific DNA binding protein are annealed and ligated to give oligomers. This DNA is then covalently coupled to Sepharose CL-2B with cyanogen bromide to yield the affinity resin. A partially purified protein fraction is combined with competitor DNA and subsequently passed through the DNA-Sepharose resin. The desired sequence-specific DNA binding protein is purified because it preferentially binds to the recognition sites in the affinity resin rather than to the nonspecific competitor DNA in solution. For example, a protein fraction that is enriched for transcription factor Sp1 can be further purified 500- to 1000-fold by two sequential affinity chromatography steps to give Sp1 of an estimated 90% homogeneity with 30% yield. In addition, the use of tandem affinity columns containing different protein binding sites allows the simultaneous purification of multiple DNA binding proteins from the same extract. This method provides a means for the purification of rare sequence-specific DNA binding proteins, such as Sp1 and CAAT-binding transcription factor.
A large region (approximately equal to 95 kDa) of human factor VIII is dispensable for in vitro procoagulant activity.
Authors: Toole J J , Pittman D D , Orr E C , Murtha P , Wasley L C , Kaufman R J
Published: May 2006

Factor VIII (antihemophilic factor) is a high molecular weight plasma glycoprotein that participates in the blood clotting cascade. The recent cloning and sequence analysis of the cDNA encoding human factor VIII revealed an obvious domain structure for the protein, which can be represented as A1-A2-B-A3-C1-C2. We now report the DNA sequence analysis of porcine exons encoding the entire B domain and part of the A2 and A3 domains. We found an unusually high degree of porcine-human amino acid sequence divergence in the B region compared with the limited sequence available for other regions of the porcine factor VIII molecule. In addition to sequence divergence, there are numerous gaps in the porcine B domain totalling over 200 amino acids. Recombinant DNA techniques were used to effect the removal of large segments of DNA encoding the B domain from the full-length human factor VIII cDNA. These constructs directed the synthesis of biologically active factor VIII when introduced into mammalian cells despite the deletion of up to 38% of the factor VIII molecule.
Transient hypoxia enhances the frequency of dihydrofolate reductase gene amplification in Chinese hamster ovary cells.
Authors: Rice G C , Hoy C , Schimke R T
Published: May 2006

Exposure of Chinese hamster cells to reduced oxygen partial pressure results in a marked enhancement in the frequency of methotrexate resistance and dihydrofolate reductase gene amplification. The frequency of enhanced resistance is a function of the length of exposure to hypoxic conditions and the time after recovery from hypoxia when cells are plated into methotrexate-containing medium. Hypoxia results in an inhibition of DNA synthesis; upon return to normal oxygen atmosphere, greater than 60% of cells in S phase at the time hypoxia was started subsequently undergo overreplication of DNA within a single cell cycle. The cells with the increased frequency of gene amplification are derived from this subset of overreplicated cells. These results are discussed within the context of the hypoxic state of many solid tumors and the high frequency of aneuploidy, chromosomal aberrations, and spontaneously occurring resistances to a number of cancer chemotherapeutic agents.
Isolation of point mutations in bacteriophage Mu attachment regions cloned in a lambda::mini-Mu phage.
Authors: Burlingame R P , Obukowicz M G , Lynn D L , Howe M M
Published: May 2006

Twenty-one derivatives of a lambda::mini-Mu phage containing point mutations in the Mu attachment regions were isolated after mutD mutagenesis and selection for relief from Mu-specific replicative interference of lambda growth. DNA sequence analysis revealed that the single left-end mutant had suffered a T----C transition at position 1 of the Mu sequence, while the remaining 20 right-end mutants contained single base-pair insertions or deletions within the terminal 19 base pairs. A genetic assay showed that the right-end mutations revealed by sequencing were necessary for relief of the replicative inhibition of lambda growth. The properties of these mutants suggest that the terminal 2-base-pair and subterminal 8-base-pair inverted repeats are important for Mu-specific replicative transposition.
Introduction of plasmid DNA into the trypanosomatid protozoan Crithidia fasciculata.
Authors: Hughes D E , Simpson L
Published: May 2006

Crithidia fasciculata cells were treated with a plasmid (pDK96) containing pBR322 sequences, a Leishmania tarentolae maxicircle autonomously replicating sequence, and the bacterial gene for aminoglycoside 3' phosphotransferase I inserted between the yeast alcohol dehydrogenase 1 promotor and terminator sequences. Resistant colonies were selected on agar plates containing paromomycin and screened for vector DNA by hybridization. Approximately 1% of the resistant colonies contained detectable vector DNA, which was present as extrachromosomal closed circular molecules ranging in copy number from 1 to 160 per cell. The plasmids could be recovered from Escherichia coli transformed to ampicillin resistance with Crithidia total cell DNA. Most of the recovered plasmids were a deleted product of pDK96, which lacked the maxicircle autonomously replicating sequence and contained a unique fragment of Crithidia nuclear DNA present at a low copy number in the wild-type genome. The plasmid DNA in resistant Crithidia was unstable even under selective conditions and was lost within 30 cell divisions.
Tyrosine 3-hydroxylase in rat brain and adrenal medulla: hybridization histochemistry and immunohistochemistry combined with retrograde tracing.
Authors: Schalling M , Hökfelt T , Wallace B , Goldstein M , Filer D , Yamin C , Schlesinger D H
Published: May 2006

Rat brain and adrenal gland were analyzed by hybridization histochemistry using an RNA probe complementary to mRNA for tyrosine 3-hydroxylase (TyrOHase; tyrosine 3-monooxygenase, EC 1.14.16.2), by immunohistochemistry using TyrOHase antiserum, and by retrograde tracing using the fluorescent compound Fast blue. Cell bodies in the ventral mesencephalon contained mRNA for TyrOHase, and these cells were also TyrOHase immunoreactive. After injection of Fast blue into the striatum, such double-labeled cells in addition contained the retrograde tracer, showing that these cells send axonal projections to the injection site. These results show that hybridization histochemistry can be used to identify transmitter-specific neuron populations and that their projections can be established.
Allosteric interpretation of the measurement of cooperative free energy in cyanomethemoglobin.
Authors: Ferrone F A
Published: May 2006

The experimentally resolved cooperative energies in partially ligated cyanomethemoglobin [F. R. Smith & G. K. Ackers (1985) Proc. Natl. Acad. Sci. USA 82, 5347-5351] have been compared with the predictions of an allosteric description of hemoglobin. A pattern of energetics similar to that observed (a "combinatorial switch") arises naturally from such an analysis using parameters in excellent agreement with other determinations. Although the energies for 2 of the 10 ligation states (namely, doubly ligated asymmetric tetramers) differ from the predictions, the remaining 8 of the 10 states exhibit excellent quantitative agreement with an allosteric description. This explains the discrepancy between previous analyses, which had found cyanomethemoglobin to be allosteric, and provides support for the basic allosteric concept that quaternary structure is the primary modifier for ligand affinity in hemoglobin.
Xylosylated-proteoglycan-induced Golgi alterations.
Authors: Kanwar Y S , Rosenzweig L J , Jakubowski M L
Published: May 2006

The effect of p-nitrophenyl beta-D-xylopyranoside on the Golgi apparatus and proteoglycans (PG) of the renal glomerulus was investigated in an isolated kidney organ perfusion system and monitored by utilizing [35S]sulfate as the PG precursor. By electron microscopy, a selective intracytoplasmic vesiculization of Golgi apparatus of visceral epithelium was observed in the beta-xyloside-treated kidneys. Electron microscopic autoradiography revealed most grains localized to the intracytoplasmic Golgi-derived vesicles, while very few grains were associated with the extracellular matrix membranes. Biochemically, a 2.3-fold increase in cellular matrix and a reduction by a factor of 1.7 in extracellular matrix of [35S]sulfate incorporation was observed. Besides a larger macromolecular form (Kavg = 0.25; Mr = 130,000), lower molecular weight PGs were recovered in the cellular (Kavg = 0.46, Mr = 30,000) and matrical (Kavg = 0.42, Mr = 45,000) compartments after xyloside treatment. The xyloside treatment increased the incorporated radioactivity, mostly included in free glycosaminoglycans and small PGs, in the media fraction by 3.8-fold. These data indicate that xyloside induces a dramatic imbalance in the de novo-synthesized PGs of cellular and extracellular compartments and that cellular accumulation of xylosylated (sulfated) PGs selectively alters the Golgi apparatus of the glomerular epithelial cell, the cell that actively synthesizes PGs.
Evolution of the silk moth chorion gene superfamily: gene families CA and CB.
Authors: Lecanidou R , Rodakis G C , Eickbush T H , Kafatos F C
Published: May 2006

The nucleotide sequences of two developmentally early chorion cDNA clones from Bombyx mori define two distinct proline-rich chorion protein families, which we name CA and CB to indicate their homologies to the previously defined chorion protein families A and B, as well as the developmentally late and cysteine-rich HcA and HcB chorion families. Thus, the chorion gene superfamily has two symmetrical branches, each consisting of three families: the alpha branch (A, CA, HcA families) and the beta branch (B, CB, HcB families). The evolution of the superfamily is discussed.
Purified human platelet-derived growth factor receptor has ligand-stimulated tyrosine kinase activity.
Authors: Bishayee S , Ross A H , Womer R , Scher C D
Published: May 2006

The platelet-derived growth factor receptor (PDGF-R), a 180-kDa single-chain polypeptide, was purified from membranes of the human osteogenic sarcoma cell line MG-63. Purification was achieved by treatment of membranes with PDGF and ATP, followed by solubilization with nonionic detergent and successive chromatography on solid-phase anti-phosphotyrosine monoclonal antibody and DEAE-cellulose. The PDGF-R, which was estimated to be 50-80% pure by NaDodSO4/polyacrylamide gel electrophoresis of 32P-labeled preparations, was free of contaminating epidermal growth factor receptor and had no detectable phosphatase activity. It specifically bound 125I-labeled PDGF, a reaction quantified by binding of the ligand-PDGF-R complex to the anti-phosphotyrosine antibody. The purified receptor displayed PDGF-stimulatable tyrosine kinase activity, assayed by autophosphorylation of PDGF-R at tyrosine residues and by phosphorylation of angiotensin II. The Km for ATP in the autophosphorylation reaction was 7.5 microM. Addition of PDGF did not change the Km but increased the Vmax 1.7-fold.
Cloning of a cDNA coding for human factor V, a blood coagulation factor homologous to factor VIII and ceruloplasmin.
Authors: Kane W H , Davie E W
Published: May 2006

Coagulation factor V is a high molecular weight plasma glycoprotein that participates as a cofactor in the conversion of prothrombin to thrombin by factor Xa. A phage lambda gt11 Hep G2 cell cDNA expression library was screened by using an affinity-purified antibody to human factor V, and 11 positive clones were isolated and plaque-purified. The clone containing the largest cDNA insert contained 2970 nucleotides and coded for 938 amino acids, a stop codon, and 155 nucleotides of 3' noncoding sequence including a poly(A) tail. The coding region includes 651 amino acids from the carboxyl terminus that constitute the light chain of human factor Va and 287 amino acids that are part of the connecting region of the protein. The predicted amino acid sequence agreed completely with 147 amino acid residues that were identified by Edman degradation of cyanogen bromide peptides isolated from the light chain. During the activation of factor V, several peptide bonds are cleaved by thrombin, giving rise to a heavy chain, a connecting fragment(s), and a light chain. The light chain is generated by the cleavage of an Arg-Ser peptide bond. The amino acid sequence of the light chain is homologous (40%) with the carboxyl-terminal fragment (Mr, 73,000) of human factor VIII. Both fragments have a similar domain structure that includes a single ceruloplasmin-related domain followed by two C domains. The carboxyl terminus of the connecting region, however, shows no significant amino acid sequence homology with factor VIII. It is very acidic and contains a number of potential N-linked glycosylation sites. It also contains about 20 tandem repeats of nine amino acids.
Proton percolation on hydrated lysozyme powders
Authors: Careri G. , Giansanti A. , Rupley John A.
Published: May 2006

The framework of percolation theory is used to analyze the hydration dependence of the capacitance measured for protein samples of pH 3-10, at frequencies from 10 kHz to 4 MHz. For all samples there is a critical value of the hydration at which the capacitance sharply increases with increase in hydration level. The threshold
h
c
= 0.15 g of water per g of protein is independent of pH below pH 9 and shows no solvent deuterium isotope effect. The fractional coverage of the surface at
h
c
is in close agreement with the prediction of theory for surface percolation. We view the protonic conduction process described here for low hydration and previously for high hydration as percolative proton transfer along threads of hydrogen-bonded water molecules. A principal element of the percolation picture, which explains the invariance of
h
c
to change in pH and solvent, is the sudden appearance of long-range connectivity and infinite clusters at the threshold
h
c
. The relationship of the protonic conduction threshold to other features of protein hydration is described. The importance of percolative processes for enzyme catalysis and membrane transport is discussed.
Human platelets contain and secrete osteonectin, a major protein of mineralized bone.
Authors: Stenner D D , Tracy R P , Riggs B L , Mann K G
Published: May 2006

We have developed a solid-phase competitive radioimmunoassay for human osteonectin, using a monoclonal antibody to bovine osteonectin. The assay is both specific and sensitive, being capable of measuring as little as 10 ng of osteonectin. Osteonectin measurements in parallel serum and plasma samples obtained from healthy individuals showed the plasma level to be 0.9 microgram/ml, while that of serum was 3 times higher, 2.6 micrograms/ml. Radioimmunoassay of blood cells indicated that platelets contain osteonectin at 1.9 micrograms per 2 X 10(8) cells. Further, the protein is released after thrombin stimulation of these cells. Immunoblot analyses of washed pelleted human platelets resulted in the identification of a single immunoreactive species. The molecular weight of this immunoreactive species was identical to that obtained for purified bovine bone osteonectin. The isolation procedure developed for bovine bone osteonectin was applied to human platelets and bone. The individual steps of the isolation procedure yielded identical profiles of immunoreactive material for bone and platelet extracts. Results of reverse-phase high-pressure liquid chromatography of bone- and platelet-derived osteonectin are consistent with the conclusion that the two sources yield the identical protein.
A role for T3+4-6-8- transitional thymocytes in the differentiation of mature and functional T cells from human prothymocytes.
Authors: Toribio M L , Martinez C , Marcos M A , Marquez C , Cabrero E , de la Hera A
Published: May 2006

In vivo, immunocompetent T lymphocytes are only detected late in ontogeny, among mature thymocytes expressing either T4 (L3T4 in mouse) or T8 (Lyt-2) surface glycoproteins. We have previously shown, however, that there are functional precursors among T3+4-6-8- human thymocytes in vivo. Here we report on the in vitro differentiation of prothymocytes into T3+4-6-8- and mature T cells. T11+3-4-6-8- prothymocytes (0.5% of total thymocytes, greater than 98% pure) were obtained after treatment of thymocytes with OKT3 (T3), OKT4A (T4), Na1/34 (T6), and B9.4 (T8) monoclonal antibodies plus complement. During culture, the prothymocyte precursors acquire first T3 and then either T4 or T8, but not T6. The largest subpopulation in the thymus, T4+6+8+ cells, are not detected among the in vitro T-cell precursors. During culture, the precursors acquire cytolytic activity as soon as they express either the T3+4-6-8- or the mature (T3+4+8- or T3+4-8+) phenotypes. We suggest that T3+4-6-8- cells are a productive, transitional stage in T-lymphocyte development.
Antitumor effects of L6, an IgG2a antibody that reacts with most human carcinomas.
Authors: Hellström I , Beaumier P L , Hellström K E
Published: May 2006

Mouse monoclonal antibody L6 (IgG2a subtype) recognizes a ganglioside antigen expressed at the surface of cells from human non-small-cell lung carcinomas, breast carcinomas, and colon carcinomas. We now show that this antibody can lyse L6 antigen-positive human tumor cells in the presence of Leu-11b-positive human lymphocytes (i.e., mediate antibody-dependent cellular cytotoxicity) or human serum (mediate complement-dependent cytotoxicity) and that it can inhibit the outgrowth of an L6 antigen-positive human tumor transplanted onto nude mice.
Restoration of flagellar clockwise rotation in bacterial envelopes by insertion of the chemotaxis protein CheY.
Authors: Ravid S , Matsumura P , Eisenbach M
Published: May 2006

When cells of the bacterium Salmonella typhimurium are incubated with penicillin and lysed in a dilute buffer, flagellated cytoplasm-free envelopes are formed. When the envelopes are tethered to glass by their flagella and then energized, some of them spin. The direction of rotation of wild-type envelopes is exclusively counterclockwise (CCW). We perturbed this system by including in the lysis medium (and hence in the envelopes) the chemotaxis protein CheY. As a result, some of the envelopes rotated exclusively clockwise (CW). The fraction of envelopes that did so increased with the concentration of CheY; at a concentration of 48 microM (pH 8), all functional envelopes spun CW. The fraction also increased with the pH of the lysis medium in the range of 6.6-8.4. The results were the same in the presence or absence of intracellular Ca2+. Reconstituted envelopes failed to respond to chemotactic stimuli. None of them changed the direction of their rotation. However, when the intracellular pH was lowered to 6.6 or below, envelopes that spun CW stopped rotating, while envelopes that spun CCW continued to rotate. This phenomenon was reversible. We conclude that CheY per se, without any additional free cytoplasmic mediators, interacts with a switch at the base of the flagellum to cause CW rotation.
Color discrimination in halobacteria: spectroscopic characterization of a second sensory receptor covering the blue-green region of the spectrum.
Authors: Wolff E K , Bogomolni R A , Scherrer P , Hess B , Stoeckenius W
Published: May 2006

Halobacterium halobium is attracted by green and red light and repelled by blue-green and shorter wavelength light. a photochromic, rhodopsin-like protein in the cell membrane, sensory rhodopsin sR587, has been identified as the receptor for the long-wavelength and near-UV stimuli. Discrepancies between the action spectrum for the repellent effect of blue light and the absorption spectrum of sR587 and its photocycle intermediate S373 strongly suggest the existence of an additional photoreceptor for the blue region of the spectrum. Transient light-induced absorbance changes in intact cells and cell membranes show, in addition to sR587, the presence of a second photoactive pigment with maximal absorption near 480 nm. It undergoes a cyclic photoreaction with a half-time of 150 msec. One intermediate state with maximal absorption near 360 nm has been resolved. The spectral properties of the new pigment are consistent with a function as the postulated photoreceptor for the repellent effect of blue light. The phototactic reactions and both pigments are absent when retinal synthesis is blocked; both can be restored by the addition of retinal. These results confirm and extend similar observations by Takahashi et al. [Takahashi, T., Tomioka, H., Kamo, N. & Kobatake, Y. (1985) FEMS Microbiol. Lett. 28, 161-164]. The archaeobacterium H. halobium thus uses two different mechanisms for color discrimination; it uses two rhodopsin-like receptors with different spectral sensitivities and also the photochromicity of at least one of these receptors to distinguish between three regions covering the visible and near-UV spectrum.
Production of platelet-derived growth factor-like molecules by cultured arterial smooth muscle cells accompanies proliferation after arterial injury.
Authors: Walker L N , Bowen-Pope D F , Ross R , Reidy M A
Published: May 2006

The migration and proliferation of smooth muscle cells (SMCs) within the intima of arteries following mechanical injury is thought to be initiated by vessel wall injury and release of growth factors, in particular the platelet-derived growth factor (PDGF). However, the mechanism by which SMC proliferation is regulated after platelet interaction with the vessel wall has ceased is unknown. Here we show that SMCs derived from the intima of injured rat arteries (intimal SMCs) are phenotypically distinct from SMCs from unmanipulated vessels (medial SMCs). Intimal SMCs secrete 5-fold greater amounts of PDGF-like activity into conditioned medium in culture, have fewer receptors for 125I-labeled PDGF, and are not mitogenically stimulated by exogenous purified PDGF. This study demonstrates that two SMC phenotypes can develop in the adult rat artery and suggests that SMC proliferation in vivo may be controlled, in part, by SMCs that produce PDGF-like molecules.
Transformation of Phycomyces blakesleeanus to G-418 resistance by an autonomously replicating plasmid.
Authors: Revuelta J L , Jayaram M
Published: May 2006

The fungus, Phycomyces blakesleeanus, shows many well-defined responses to a number of external stimuli. Genetic analysis shows that at least eight genes are involved in Phycomyces sensory transduction. As a first step toward the molecular analysis of these genes and their products, we have developed a transformation protocol for Phycomyces by using a plasmid containing the kanamycin-resistance gene from Tn903 and a Phycomyces DNA fragment capable of supporting autonomous replication in yeast (ARS). Our results demonstrate that the Tn903 gene is expressed in Phycomyces and that the ARS fragment selected in yeast supports autonomous replication in Phycomyces as well.
Two mechanisms for directional gene conversion.
Authors: Hamza H , Kalogeropoulos A , Nicolas A , Rossignol J L
Published: May 2006

G234 is a large silent deletion located in the middle of gene b2, which controls spore pigmentation in Ascobolus immersus. Its gene conversion directionality was studied in asci, which show evidence of heteroduplex DNA at flanking markers, and was compared to the behavior of closely linked single-base-pair insertions or deletions. We found that with the G234 deletion, the genotype of the donor strand in the heteroduplex is preferentially recovered, irrespective of its G234 or wild-type nature, whereas with single-base-pair insertions or deletions, the direction of conversion favors one genotype, whether it was the donor or the recipient strand. We conclude that there exists two mechanisms for directional gene conversion, the "donor-directed" conversion mechanism being epistatic to the "genotype-directed" one. We discuss these data with regard to models for mismatch repair.
AKR murine thymic leukemias are from a distinct thymic cell lineage and do not express the beta chain of the T-cell antigen receptor.
Authors: Owen F L , Strauss W M , Murre C , Duby A D , Hiai H , Seidman J G
Published: May 2006

Characterization of tumors that arise spontaneously in the AKR mouse indicates that they are derived from cells of a distinct T-cell lineage. Cells in this subclass bear surface antigens, designated Tpre, Tthy, Tind, and Tsu, which are encoded by genes in the Tsu linkage group on murine chromosome 12. We have examined the rearrangement and expression of genes encoding the T-cell alpha, beta, and gamma chains in these tumors. Although these cells contain alpha-chain mRNA, they do not produce a normal-sized beta-chain mRNA. Most of them also lack gamma-chain mRNA. Each thymic leukemia was derived from a cell arrested at a different stage of development as defined by their expression of terminal deoxynucleotidyl transferase and Thy-1 mRNA. The data presented here are consistent with a model in which thymocytes expressing Tpre, Tthy, Tind, or Tsu undergo somatic development parallel to the development of other T cells. However, these thymocytes do not appear to differentiate into cells bearing alpha-beta heterodimers of the T-cell antigen receptor.
Epoxidation of (+/-)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene during (bi)sulfite autoxidation: activation of a procarcinogen by a cocarcinogen.
Authors: Reed G A , Curtis J F , Mottley C , Eling T E , Mason R P
Published: May 2006

The (bi)sulfite ion undergoes extensive autoxidation in neutral aqueous media with the formation of sulfur trioxide radical anion that is detected by ESR. The radical anion subsequently reacts with molecular oxygen to form a peroxyl radical. We find that when (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) is included in this autoxidation system, BP-7,8-diol is converted to diolepoxides, ultimate carcinogenic derivatives of benzo[a]pyrene. This epoxidation occurs with a stereoselectivity consistent with either a peroxyl radical or a peracid as the epoxidizing agent. The epoxidation is dependent on the concentration of both (bi)sulfite and oxygen. In the presence of 10 microM butylated hydroxyanisole, which abolishes (bi)sulfite autoxidation, no (bi)sulfite-dependent epoxidation occurs. These results are discussed in regard to the mechanism of (bi)sulfite autoxidation, and in relationship to the cocarcinogenicity of sulfur dioxide [anhydrous (bi)sulfite] for benzo[a]pyrene-induced pulmonary neoplasia.
Phosphorylation and inactivation of protein phosphatase 1 by pp60v-src.
Authors: Johansen J W , Ingebritsen T S
Published: May 2006

Protein phosphatase 1, one of four major protein phosphatases involved in cellular regulation, was phosphorylated in vitro by pp60v-src, the transforming gene product of Rous sarcoma virus. Phosphorylation was accompanied by a loss of protein phosphatase activity. The inactivation of protein phosphatase 1 was time-dependent and the extent of inactivation correlated closely with the stoichiometry of phosphorylation. Under optimal conditions, 0.34 +/- 0.01 mol of phosphate were incorporated per mol of protein phosphatase and the activity of the enzyme was decreased by 39 +/- 2%. The inactivation required the presence of both MgATP and pp60v-src. There was no loss of activity when adenosine 5'-[beta gamma-imido]triphosphate was used in place of ATP. Phosphorylation of protein phosphatase 1 occurred exclusively on tyrosine residues and was blocked by specific antibodies to pp60v-src. During preincubation of pp60v-src at 41 degrees C, its protein kinase activity towards casein was lost rapidly. The ability of pp60v-src to phosphorylate and inactivate protein phosphatase 1 declined in parallel with the loss of casein kinase activity. Limited chymotryptic digestion of 32P-labeled protein phosphatase 1 (Mr 37,000) resulted in its quantitative conversion to a Mr 33,000 species. Conversion to this species was accompanied by the loss of 32P-labeling and by reactivation of the protein phosphatase. When various concentrations of chymotrypsin were used in the digestion, there was a close correlation between conversion to the Mr 33,000 species and the restoration of protein phosphatase activity. pp60v-src was unable to phosphorylate or inactivate a partially proteolyzed species of protein phosphatase 1 (Mr 33,000/34,000).
Antigenic determinants in proteins coincide with surface regions accessible to large probes (antibody domains).
Authors: Novotný J , Handschumacher M , Haber E , Bruccoleri R E , Carlson W B , Fanning D W , Smith J A , Rose G D
Published: May 2006

We evaluated surface areas on proteins that would be accessible to contacts with large (1-nm radius) spherical probes. Such spheres are comparable in size to antibody domains that contain antigen-combining sites. We found that all the reported antigenic sites correspond to segments particularly accessible to a large sphere. The antigenic sites were also evident as the most prominently exposed regions (hills and ridges) in contour maps of the solvent-accessible (small-probe) surface. In myoglobin and cytochrome c, virtually all of the van der Waals surface is accessible to the large probe and therefore potentially antigenic; in myohemerythrin, distinct large-probe-inaccessible, and nonantigenic, surface regions are apparent. The correlation between large-sphere-accessibility and antigenicity in myoglobin, lysozyme, and cytochrome c appears to be better than that reported to exist between antigenicity and segmental flexibility; that is, surface regions that are rigid often constitute antigenic epitopes, whereas some of the flexible parts of the molecules do not appear antigenic. We propose that the primary reason why certain polypeptide-chain segments are antigenic is their exceptional surface exposure, making them readily available for contacts with antigen-combining sites. Exposure of these segments frequently results in high mobility and, in consequence, to the reported correlation between antigenicity and segmental flexibility.
Mechanism of the idling-turnover reaction of the large (Klenow) fragment of Escherichia coli DNA polymerase I.
Authors: Mizrahi V , Benkovic P A , Benkovic S J
Published: May 2006

The mechanism of the idling-turnover reaction catalyzed by the large (Klenow) fragment of Escherichia coli DNA polymerase I has been investigated. The reaction cycle involved is one of excision/incorporation, in which the 3' deoxynucleotide residue of the primer DNA strand is partitioned into its 5'-mono- and 5'-triphosphate derivatives, respectively. Mechanistic studies suggest the 5'-monophosphate product is formed in the first step by simple 3'----5' exonucleolytic cleavage. Rapid polymerization follows with the concomitant release of inorganic pyrophosphate. In the second step, the 5'-triphosphate product is generated by a pyrophosphorolysis reaction, which, despite the low concentration of pyrophosphate that has accumulated, occurs at a rate that is comparable with that of the parallel 3'----5' hydrolysis reaction.
Antisera of designed specificity for subunits of guanine nucleotide-binding regulatory proteins.
Authors: Mumby S M , Kahn R A , Manning D R , Gilman A G
Published: May 2006

Antisera were raised against purified subunits of regulatory GTP-binding proteins (G proteins) and against synthetic peptides that correspond to defined regions of G proteins. Peptide antisera were generated that recognized all alpha or all beta subunits from Gs, Gi, Go, and transducin; others recognized only Gs alpha or Go alpha. Such cross-reaction or complete specificity for a given alpha subunit was not obtained when purified subunits were injected. Peptide antisera were used to identify G protein subunits in selected tissue membrane preparations by immunoblots.
Upstream sequences confer distinctive transcriptional properties on genes encoding silkgland-specific tRNAAla.
Authors: Young L S , Takahashi N , Sprague K U
Published: May 2006

To understand the molecular basis for the tissue-specific accumulation of alanine tRNAs in silkworms, we have compared the transcriptional properties of genes that encode constitutive and silkgland-specific type alanine tRNA. Genes for each class of alanine tRNA behave very differently when used as templates for homologous in vitro transcription. Since these properties are likely related to the activities of the corresponding genes in vivo, we wished to identify the cis-acting elements responsible for them. We have therefore constructed hybrid silkgland-specific/constitutive genes and have analyzed their capacity to direct transcription in vitro. We find a simple pattern: the distinctive transcriptional properties of the two kinds of tRNAAla genes are the result of different positive signals upstream from their sites of transcription initiation.
Molecular characterization of the Drosophila vermilion locus and its suppressible alleles.
Authors: Searles L L , Voelker R A
Published: May 2006

We have cloned vermilion (v), one of the genes required for brown eye pigment synthesis in Drosophila, using a mutant (vH2a) "tagged" with the transposon P factor. Mutations that disrupt v gene expression are clustered within approximately 2 kilobases of DNA. A 1.4-kilobase transcript, homologous to this same region, is present in v+ RNA but absent in RNA from several v mutants. The spontaneous v alleles that are suppressed by the suppressor of sable [su(s)] are apparently identical insertions of 412, a copia-like transposable element. Preliminary evidence suggests that su(s)-suppressible alleles at other loci may also be 412 insertions.
Evidence for rearrangement, amplification, and expression of c-myc in a human glioblastoma.
Authors: Trent J , Meltzer P , Rosenblum M , Harsh G , Kinzler K , Mashal R , Feinberg A , Vogelstein B
Published: May 2006

Amplified cellular genes are frequently manifested in one of two cytologically recognizable forms, double minutes or homogeneously staining regions. Additionally, evidence is accumulating that aberrant expression of cellular genes (including oncogenes) may be mediated by gene amplification. We now describe the amplification and expression of the cellular oncogene c-myc in double-minute-containing cells from a patient with glioblastoma multiforme, and we have shown that the amplification is associated with rearrangement of the c-myc gene. This finding further supports the common association of the myc gene family in neurogenic tumors and provides evidence of myc gene amplification in human brain cancer.
Monte Carlo estimation of the electric field in stellarators
Authors: Bauer F. , Betancourt O. , Garabedian P. , Ng K. C.
Published: May 2006

The BETA computer codes have been developed to study ideal magnetohydrodynamic equilibrium and stability of stellarators and to calculate neoclassical transport for electrons as well as ions by the Monte Carlo method. In this paper a numerical procedure is presented to select resonant terms in the electric potential so that the distribution functions and confinement times of the ions and electrons become indistinguishable.
X-ray crystallographic investigation of substrate binding to carboxypeptidase A at subzero temperature.
Authors: Christianson D W , Lipscomb W N
Published: May 2006

A high-resolution x-ray crystallographic investigation of the complex between carboxypeptidase A (CPA; peptidyl-L-amino-acid hydrolase, EC 3.4.17.1) and the slowly hydrolyzed substrate glycyl-L-tyrosine was done at -9 degrees C. Although this enzyme-substrate complex has been the subject of earlier crystallographic investigation, a higher resolution electron-density map of the complex with greater occupancy of the substrate was desired. All crystal chemistry (i.e., crystal soaking and x-ray data collection) was performed on a diffractometer-mounted flow cell, in which the crystal was immobilized. The x-ray data to 1.6-A resolution have yielded a well-resolved structure in which the zinc ion of the active site is five-coordinate: three enzyme residues (glutamate-72, histidine-69, and histidine-196) and the carbonyl oxygen and amino terminus of glycyl-L-tyrosine complete the coordination polyhedron of the metal. These results confirm that this substrate may be bound in a nonproductive manner, because the hydrolytically important zinc-bound water has been displaced and excluded from the active site. It is likely that all dipeptide substrates of carboxypeptidase A that carry an unprotected amino terminus are poor substrates because of such favorable bidentate coordination to the metal ion of the active site.
Trans-activation of a methylated adenovirus promoter by a frog virus 3 protein.
Authors: Thompson J P , Granoff A , Willis D B
Published: May 2006

The high degree of methylation of the frog virus 3 (FV3) genome suggests that FV3-infected cells are capable of transcribing highly methylated DNA. We tested this hypothesis by assaying the transcriptional activity of adenovirus promoters known to be inhibited by methylation. Plasmid constructs containing the E1a and E2aE promoters of adenovirus type 12 linked to the gene for chloramphenicol acetyltransferase [(CAT) EC 2.3.1.28], when methylated and introduced into eukaryotic cells, promoted CAT synthesis only when the cells were subsequently infected with FV3. Mapping of transcriptional initiation sites revealed that the same sites in the E1a promoter were used for the initiation of transcription in uninfected and infected cells. Moreover, Southern blots showed that transfected plasmid DNA from FV3-infected cells was not demethylated. The absence of CAT-specific RNA in transfected cells infected with FV3 in the presence of protein synthesis inhibitors demonstrated that a virus-induced protein was responsible for the trans-activation. Inhibition of transcription from the methylated template by alpha-amanitin indicated that a functional host RNA polymerase II is required for transcription of methylated DNA in FV3-infected cells. The virus-induced trans-acting protein presumably alters either host RNA polymerase II or the methylated DNA template to allow transcription from the methylated adenovirus promoters.
Isolation of the origin of replication associated with the amplified Chinese hamster dihydrofolate reductase domain.
Authors: Burhans W C , Selegue J E , Heintz N H
Published: May 2006

Autoradiography of restriction digests of DNA labeled in early S phase indicates that replication of the amplified dihydrofolate reductase (DHFR) domain of methotrexate-resistant CHOC 400 cells initiates within a 6.1-kilobase pair (kb) EcoRI-doublet located on the 3' side of the DHFR gene. To localize the DHFR origin fragment, synchronized CHOC 400 cells were either pulse labeled with [3H]thymidine in vivo or permeabilized and incubated with [32P]dATP under conditions that support limited chromosomal DNA replication. The temporal order of replication of amplified fragments was determined by hybridization of the in vivo or in vitro replication products to cloned fragments spanning the earliest-replicating portion of the DHFR domain. At the G1/S boundary, the labeled products derived from the replication of amplified sequences, either in whole or permeabilized cells, are distributed about an amplified 4.3-kb Xba I fragment that maps 14 kb downstream from the DHFR gene. As cells progress through the S phase, bidirectional replication away from this site is observed. These studies indicate that the 4.3-kb Xba I fragment contains the origin of replication associated with the amplified DHFR domain.